Antigenic chimeric tick-borne encephalitis virus/dengue virus type 4 recombinant viruses

ABSTRACT

Disclosed herein are chimeric TBEV/DEN4 flaviviruses including a first nucleic acid molecule including a 5′ non-coding region (NCR) from a DEN4 virus, a nucleic acid encoding a C protein and non-structural proteins from a DEN4 virus, and a 3′ NCR from a DEN4 virus, wherein nonstructural protein NS4B includes a phenylalanine at amino acid position 112, nonstructural protein NS5 includes an alanine at amino acid position 654 and an alanine at amino acid position 655, and the 3′ NCR includes a deletion of nucleotides 10478-10507. The chimeric construct also includes a second nucleic acid molecule, which is operably linked to the first nucleic acid molecule, encoding a prM protein and an E protein from a TBEV, wherein the E protein includes an amino acid substitution that differs from the wild type TBEV at amino acid position 315 and a tryptophan at amino acid position 240. Also disclosed are methods of eliciting an immune response using the disclosed TBEV/DEN4 chimeric flaviviruses and immunogenic compositions including the disclosed chimeric flaviviruses and a pharmaceutically acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATION

This is the U.S. National Stage of International Application No. PCT/US2010/036678, filed May 28, 2010, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Patent Application Ser. No. 61/181,982, filed May 28, 2009, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to chimeric flaviviruses, particularly chimeric tick-borne encephalitis virus/dengue virus type 4 chimeras. Further, it relates to methods of eliciting an immune response to tick-borne encephalitis virus in a host utilizing the disclosed chimeric flaviviruses.

BACKGROUND

The tick-borne encephalitis virus (TBEV) complex is a group of viruses that cause severe neurotropic disease and up to 30% mortality. While these viruses can be found in many parts of the world, the largest impact of disease occurs in Europe and Russia, where approximately 14,000 hospitalized TBEV cases occur annually. Furthermore, the majority of TBEV cases are considered to be subclinical, indicating a higher incidence of TBEV infection than is generally recognized. The tick-borne encephalitis complex includes TBEV (e.g., European, Siberian, and Far Eastern subtypes), as well as Omsk hemorrhagic fever, Kyasanur forest disease, Langat, Louping ill, Negishi, and Powassan viruses.

TBEV is in the family Flaviviridae, genus flavivirus and is composed of a positive-sense single stranded RNA genome that contains 5′ and 3′ non-coding regions (NCR) and a single open reading frame encoding 10 proteins. The proteins are co- and post-translationally cleaved by viral and host proteins to derive three structural proteins (capsid (C), membrane (M), and envelope (E)), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The non-structural proteins regulate viral transcription and replication and attenuate antiviral responses, while the structural proteins compose the outer shell of the virus and are, therefore, significant effectors of host immunogenicity.

SUMMARY OF THE DISCLOSURE

Disclosed herein are recombinant chimeric TBEV/DEN4 flaviviruses including 5′ and 3′ non-coding regions, a C protein, and non-structural proteins from DEN4 virus, and a prM protein and E protein from TBEV. In particular examples, the chimeric TBEV/DEN4 virus includes a first nucleic acid molecule including a 5′ non-coding region from a DEN4 virus, a nucleic acid encoding a C protein from a DEN4 virus, non-structural proteins from a DEN4 virus, and a 3′ non-coding region from a DEN4 virus, wherein nonstructural protein NS4B includes a phenylalanine at amino acid position 112, nonstructural protein NS5 includes an alanine at amino acid position 654 and an alanine at amino acid position 655, and the 3′ non-coding region includes a deletion of nucleotides 10478-10507 (Δ30). The chimeric TBEV/DEN4 virus also includes a second nucleic acid molecule which is operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a prM protein from a TBEV and an E protein from a TBEV, and wherein the E protein includes an amino acid substitution that differs from the wild type TBEV at amino acid position 315 and a tryptophan at amino acid position 240. In one example, the chimeric TBEV/DEN4 flavivirus construct includes aspartic acid at amino acid 315 of the TBEV E protein. In particular examples, the chimeric TBEV/DEN4 flavivirus encodes a polypeptide at least 95% identical to SEQ ID NO: 2 or includes a nucleic acid molecule at least 95% identical to SEQ ID NO: 1. In further examples, the chimeric TBEV/DEN4 flavivirus construct includes additional amino acid substitutions, such as at amino acid 84 of the TBEV E protein and/or amino acid 6 of the NS1 protein of DEN4.

Further disclosed herein is a recombinant TBEV/DEN4 chimeric virus that can be used to produce pseudoinfectious or replication-deficient TBEV/DEN4 viruses. In some examples, the TBEV/DEN4 chimera lacks at least one structural protein, such as the C protein, prM protein, and/or E protein. The deleted structural proteins are provided in a separate construct. In one example, the chimeric virus includes a first nucleic acid molecule including a 5′ non-coding region from a DEN 4 virus; a nucleic acid encoding non-structural proteins from a DEN 4 virus, wherein nonstructural protein NS4B comprises a phenylalanine at amino acid position 112, and nonstructural protein NS5 comprises an alanine at amino acid position 654 and an alanine at amino acid position 655; and a 3′ non-coding region from a DEN 4 virus, wherein the 3′ non-coding region comprises a deletion of nucleotides 10478-10507; and a second nucleic acid molecule operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a prM protein from a TBEV; and an E protein from a TBEV, wherein the E protein comprises an amino acid substitution from wild-type at amino acid position 315 and a tryptophan at amino acid position 240, wherein the chimeric virus does not encode a C protein.

Also disclosed herein are methods of eliciting an immune response to TBEV in a subject, including administering a disclosed recombinant TBEV/DEN4 virus or a disclosed recombinant pseudoinfectious or replication-deficient TBEV/DEN4 virus to the subject. In addition, an immunogenic composition is disclosed, the composition including a recombinant TBEV/DEN4 virus including the disclosed nucleic acid chimeras and a pharmaceutically acceptable carrier. Methods of eliciting an immune response to TBEV or members of the TBEV complex, including administering a therapeutically effective amount of the immunogenic composition to a subject are also disclosed.

The foregoing and other features will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of graphs showing replication of TBEV/DEN4Δ30 chimeras in Vero cells, human neuroblastoma SH-SY5Y cells, or human glioblastoma LN-18 cells at various temperatures. TBEV/DENΔ30, TBEV/DEN4Δ30 chimera; TBEV/DENΔ30/E-315, TBEV/DEN4Δ30/E-K₃₁₅D chimera; TBEV/DENΔ30/NS5-654,655, TBEV/DEN4Δ30/NS5-DR_(654,655)AA chimera; TBEV/DENΔ30/E-315/NS5-654,655, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimera.

FIG. 2A is a graph showing viral replication kinetics of TBEV/DEN4Δ30 chimeras in the brains of 3 day-old Swiss mice inoculated intracerebrally (ic) with 10³ pfu of the indicated chimeric virus and harvested at the indicated days post-inoculation. TBEV/DEN4Δ30, TBEV/DEN4Δ30 chimera; TBEV/DEN4Δ30/E-315, TBEV/DEN4Δ30/E-K₃₁₅D chimera; TBEV/DEN4Δ30/N55-654,655, TBEV/DEN4Δ30/N55-DR_(654,655)AA chimera; TBEV/DEN4Δ30/E-315/NS5-654,655, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimera

FIG. 2B is a graph showing viral replication kinetics of TBEV/DEN4Δ30 chimeras in the brains of 5 day-old Swiss mice inoculated ic with 10³ pfu of the indicated chimeric virus and harvested at the indicated days post-inoculation. TBEV/DEN4Δ30, TBEV/DEN4Δ30 chimera; TBEV/DEN4Δ30/E-315, TBEV/DEN4Δ30/E-K₃₁₅D chimera; TBEV/DEN4Δ30/N55-654,655, TBEV/DEN4Δ30/NS5-DR_(654,655)AA chimera; TBEV/DEN4Δ30/E-315/NS5-654,655, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimera.

FIG. 3 is a series of digital images showing neuroinflammation in the brain of mice infected with chimeras TBEV/DEN4 (A, D, and G); TBEV/DEN4Δ30 (B, E, and H), or TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA (vΔ30/E₃₁₅/NS5; C, F, and I). Representative images of neuroinflammation in the brain on day 6 from mice ic inoculated with each virus (H&E staining) The boxed areas in A-C (20X magnification) are shown in D-F at higher magnification (40X magnification) and G-I show the boxed areas in D-F at higher magnification (100X magnification). Inflammatory foci are indicated by arrows and the dashed circle. Abbreviations: Cx, cortex; CA1, hippocampus; DG, dentate gyrus; Th, thalamus; Or, oriens layer of the hippocampus; Py, pyramidal layer of the hippocampus; rad, radiatum layer of the hippocampus.

FIG. 4 is a bar graph showing replication of the indicated viruses in the brains of 3-week-old SCID mice following intraperitoneal (ip) inoculation with 10⁵ pfu of virus. On the indicated days, three mouse brains per group were harvested and the virus titer of each mouse brain homogenate was determined by immunofocus assay on Vero cells. Mean virus titers ±SE are indicated. The limit of detection of the assay was 1.7 log₁₀ pfu/g.

FIG. 5A is a graph showing percent survival of adult Swiss mice inoculated intraperitoneally (ip) with the indicated TBEV/DEN4 chimeras followed by intracerebral (ic) challenge with 100 icLD₅₀ (1000 pfu) of TBEV/DEN4 virus four weeks later. TBEV/DEN4, TBEV/DEN4 chimera; TBEV/DEN4/E-315, TBEV/DEN4/E-K₃₁₅D chimera; TBEV/DEN4/N55-654,655, TBEV/DEN4/N55-DR_(654,655)AA chimera; TBEV/DEN4/E-315/N55-654,655, TBEV/DEN4/E-K₃₁₅D/NS5-DR_(654,655)AA chimera; mock, vehicle.

FIG. 5B is a graph showing percent survival of adult Swiss mice inoculated ip with the indicated TBEV/DEN4Δ30 chimeras followed by ic challenge with 100 icLD₅₀ of TBEV/DEN4 virus four weeks later. TBEV/DEN4Δ30, TBEV/DEN4Δ30 chimera; TBEV/DEN4Δ30/E-315, TBEV/DEN4Δ30/E-K₃₁₅D chimera; TBEV/DEN4Δ30/N55-654,655, _(TBEV/DEN)4Δ30/NS5-DR_(654,655)AA chimera; TBEV/DEN4Δ30/E-315/N55-654,655, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimera; mock, vehicle.

FIG. 6 is a scatter plot showing immunogenic response of mice inoculated with the indicated vaccines prior to challenge with wild-type TBEV strains. Mice were immunized with one or two doses (10⁵ pfu) of live attenuated TBEV/DEN4Δ30/E₃₁₅/NS5_(654,655), three doses of inactivated Encepur® vaccine, or mock vaccinated. Serum neutralizing antibody titers were measured against TBEV/DEN4Δ30. Significant differences (designated by asterisks) were observed between one and two doses of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR-_(654,655)AA (TBEV/DEN4Δ30/E₃₁₅/NS5_(654,655); p<0.05), whereas no significant differences were observed between two doses of TBEV/DEN4Δ30/E₃₁₅/NS5_(654,655) and three doses of Encepur® for neutralizing titers (p>0.05, one-way ANOVA followed by Tukey post-hoc or unpaired t test). Reciprocal mean titers are indicated by the bars.

FIG. 7 is a pair of graphs showing viral replication in simian Vero (A) or mosquito C6/36 (B) cells. Cells were infected with TBEV/DEN4Δ30 (♦), its derived mutant viruses (TBEV/DEN4Δ30/E-K₃₁₅D (□),TBEV/DEN4Δ30/NS5-DR-_(654,655)AA (▴),TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR-_(654,655)AA (x)), LGT(*), or DEN4 (Δ) viruses at an MOI of 1. Viral supernatants were taken every 24 hours post-infection for eight days, and were quantitated for viral titers. Each time point represents the mean viral titer from three replicates. Limit of detection was 2.0 log₁₀ pfu/ml.

FIG. 8 is a graph showing virus titer in Ae. aegypti mosquitoes after 14 or 21 days incubation. The limit of detection was 0.4 log₁₀/dmosquito.

FIG. 9 is a series of digital images of agarose gel electrophoresis of RT-PCR amplified cDNA from virus, showing presence of viral RNA in ticks following infection. Total RNA was isolated from a group of 25 I. scapularis larvae at 21 days post immersion with media or the various viruses. Viral RNA was detected using primers specific for the (A) positive or (B) negative sense RNA. To detect TBEV/DEN4Δ30 and its derivatives (E-K₃₁₅D, _(NS)5-DR_(654,655)AA and E-K₃₁₅D/NS5-DR_(654,655)AA), TBEV specific primers were used, whereas LGT- and DEN4-specific primers were used to detect LGT and DEN4 virus, respectively. Positive controls (+CNTRL) were total RNA isolated from virus infection in Vero cells. Molecular weight markers are displayed in the far left lanes. TBEV/DEN4Δ30, TBEV/DEN4Δ30 chimera; vΔ30/E₃₁₅, TBEV/DEN4Δ30/E-K₃₁₅D chimera; vΔ30/NS5_(654,655), TBEV/DEN4Δ30/NS5-DR_(654,655)AA chimera; vΔ30/E₃₁₅/NS5_(654,655), TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimera; mock, uninfected.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. §1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence_Listing.txt, which was created on Nov. 14, 2011, and is 87,787 bytes, which is incorporated by reference herein.

SEQ ID NO: 1 and 2 show the nucleic acid and amino acid sequence, respectively of a TBEV/DEN4Δ30 chimera including deletion of nucleotides 10478-10507 in the 3′ NCR, R240W and K315D mutations in the E protein, L112F mutation in the NS4B protein, and D654A and R655A mutations in the NS5 protein. The start and stop positions of the particular genes and proteins of the chimera are shown in Table 1.

TABLE 1 Start and stop positions of NCRs, structural proteins and nonstructural proteins in SEQ ID NOs: 1 and 2. SEQ ID NO: 1 nucleotide SEQ ID NO: 2 amino acid Region start/stop position start/stop position 5′ NCR  1-101 — C 102-458  1-119 prM 459-950 120-283 M 726-950 209-283 E  951-2438 284-779 NS1 2439-3494  780-1131 NS2A 3495-4148 1132-1349 NS2B 4149-4538 1350-1479 NS3 4539-6392 1480-2097 NS4A 6393-6842 2098-2247 NS4B 6843-7577 2248-2492 NS5  7578-10280 2493-3393 3′ NCR 10281-10633 —

SEQ ID NOs: 3-12 are primers used to amplify and sequence TBEV/DEN4 chimeric virus sequences.

SEQ ID NOs: 13 and 14 are forward and reverse primers, respectively, used to detect DEN4 positive-sense strand RNA.

SEQ ID NOs: 15 and 16 are forward and reverse primers, respectively, used to detect DEN4 negative-sense strand RNA.

SEQ ID NOs: 17 and 18 are forward and reverse primers, respectively, used to detect TBE positive-sense strand RNA.

SEQ ID NOs: 19 and 20 are forward and reverse primers, respectively, used to detect TBE negative-sense strand RNA.

SEQ ID NOs: 21 and 22 are forward and reverse primers, respectively, used to detect Langat positive-sense strand RNA.

SEQ ID NOs: 23 and 24 are forward and reverse primers, respectively, used to detect Langat negative-sense strand RNA.

SEQ ID NOs: 25 and 26 are forward and reverse primers, respectively, used to detect Langat/DEN negative-sense strand RNA.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

I. Abbreviations

-   -   AST: average survival time     -   DEN4: dengue virus type 4     -   dpi: days post-inoculation     -   dpc: days post-challenge     -   EOP: efficiency of plaque formation     -   hr: host range restriction     -   ic: intracerebral     -   ip: intraperitoneal     -   LD₅₀: 50% lethal dose     -   LGT: Langat virus     -   NCR: non-coding region     -   pfu: plaque forming units     -   sc: subcutaneous     -   TBEV: tick-borne encephalitis virus     -   TBEV/DEN4: virus with prM and E genes (or proteins) from TBEV         and remaining genome (genes, proteins, or NCRs) from DEN4     -   TBEV/DEN4Δ30: virus with prM and E genes (or proteins) from TBEV         and remaining genome (genes, proteins, or NCRs) from DEN4 with a         30 nucleotide deletion in the DEN4 3′ NCR     -   ts: temperature sensitive         II. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. All GenBank Accession Nos. mentioned herein are incorporated by reference in their entirety as of May 28, 2009. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to these light and heavy chains.

As used herein, the term “antibodies” includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab′)₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and devices of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In one embodiment, an antigen is a virus antigen, such as a flavivirus prM or E protein.

Attenuated: In the context of a live virus, such as a TBEV or DEN4 virus, the virus is attenuated if its ability to infect a cell or subject and/or its ability to produce disease is reduced (for example, eliminated). Typically, an attenuated virus retains at least some capacity to elicit an immune response following administration to an immunocompetent subject. In some cases, an attenuated virus is capable of eliciting a protective immune response without causing any signs or symptoms of infection. In particular examples, an attenuated flavivirus exhibits reduced neurovirulence and/or neuroinvasiveness in a host (such as a mouse, non-human primate, or human).

Capsid protein (C): One of three flavivirus structural proteins. The C protein forms the viral capsid, which contains the viral nucleic acid.

Chimera: A molecule (e.g., gene, transcript, or protein) composed of parts that are of different origin (such as at least two nucleic acid or amino acid sequences) that, while typically unjoined in their native state, are joined or linked to form a single continuous molecule. A chimera may include nucleic acid or amino acid molecules that are joined end-to-end (for example, the amino-terminus of one molecule is joined to the carboxyl-terminus of a second molecule) or may include one molecule that is embedded within another molecule (for example, the amino-terminus and carboxyl-terminus of the chimera are from one molecule, while an intervening sequence comes from another molecule).

A chimera may include a chimeric protein, for example a protein that is composed of amino acid sequences from more than one protein. A chimera may also include a chimeric nucleic acid composed of nucleic acid molecules from more than one source, such as a chimeric nucleic acid which encodes a chimeric protein. In some examples, a chimera may include a chimeric genome, such as a flavivirus genome, which is composed of sequences from two or more flaviviruses. For example, a chimeric flavivirus genome may comprise nucleic acid molecules from more than one flavivirus genome, such as dengue virus (such as DEN4) and tick-borne encephalitis virus (such as Far Eastern, Central European, or Siberian TBEV subtypes). In some examples, a chimeric flavivirus includes one or more nucleic acids encoding one or more proteins from a first flavivirus and one or more nucleic acids encoding one or more proteins from a second flavivirus. In particular examples, a chimeric flavivirus includes a nucleic acid molecule encoding the non-structural proteins and C protein from a DEN4 virus genome linked to a nucleic acid molecule encoding a prM protein and E protein from a TBEV genome.

Conservative substitution: A substitution of one amino acid residue in a protein for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, ideally, a flavivirus protein (such as a C, prM, E, or non-structural protein) including one or more conservative substitutions (for example no more than 2, 5, 10, 20, 30, 40, or 50 substitutions) retains the structure and function of the wild-type protein. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleic acid that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be readily selected for additional testing by infecting cells with a virus containing a variant protein and determining ability to replicate (for example as described in Example 1) or by producing virus containing a variant protein and determining its neurovirulence or neuroinvasion properties (for example, as described in Example 2).

Dengue virus: A member of the virus family Flaviviridae which is transmitted through the bite of the mosquitoes Aedes aegypti and Aedes albopictus. There are four antigenically distinct subtypes of dengue virus: dengue 1, dengue 2, dengue 3, and dengue 4. In particular examples, the dengue virus is dengue 4 virus.

The most common symptoms of dengue are high fever, severe headache, backache, joint pains, nausea and vomiting, eye pain, and rash. Dengue hemorrhagic fever is characterized by a fever that lasts from 2 to 7 days, with symptoms such as nausea, vomiting, abdominal pain, and headache. This stage is followed by hemorrhagic manifestations, such as tendency to bruise easily, nose bleed, and possibly internal bleeding. The capillaries become excessively permeable, allowing the fluid component to escape from the blood vessels. Dengue hemorrhagic fever or Dengue shock syndrome can be fatal (about 5% fatality rate).

Envelope protein (E): A flavivirus structural protein that mediates binding of flavivirus virions to cellular receptors on host cells. The flavivirus E protein is required for membrane fusion, and is the primary antigen inducing protective immunity to flavivirus infection. Flavivirus E protein affects host range, tissue tropism and viral virulence. The flavivirus E protein contains three structural and functional domains, DI-DIII. In mature virus particles the E protein forms head to tail homodimers lying flat and forming a dense lattice on the viral surface.

Host: A cell or organism which harbors another organism or biological entity, for example a parasite or a virus. In some examples, a host is a small mammal (e.g., a rodent), human, or non-human primate that can be or is infected by a tick-borne encephalitis virus (such as Far Eastern, Central European, or Siberian TBEV subtypes) or a dengue virus (such as DEN1, DEN2, DEN3, or DEN4).

Immune response: A response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen. An immune response can include any cell of the body involved in a host defense response, for example, an epithelial cell that secretes an interferon or a cytokine An immune response includes, but is not limited to, an innate immune response or inflammation.

Immunize: To render a subject protected from an infectious disease, such as by vaccination.

Immunogen: A compound, composition, or substance that is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, an “immunogenic composition” is a composition comprising an immunogen.

Isolated: An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, virus, or particle) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. Preferably, a preparation is purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.

Non-structural protein: One of the non-structural (NS) proteins of a flavivirus, which are designated NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The non-structural proteins are encoded by the portion of the flavivirus genome that is 3′ to the structural proteins. NS1 has been implicated in RNA replication and has been shown to be secreted from infected mammalian cells (Post et al., Virus Res. 18:291-302, 1991; Mackenzie et al., Virology 220:232-240, 1996; Muylaert et al., Virology 222:159-168, 1996). NS1 can elicit strong humoral immune responses and is a potential vaccine candidate (Shlesinger et al., J. Virol. 60:1153-1155, 1986; Qu et al., J. Gen. Virol. 74:89-97, 1993). The function of NS2A is unknown. NS2B forms a complex with NS3 and functions as a cofactor for the NS3 protease, which cleaves portions of the virus polyprotein. NS3 also functions as an RNA helicase and is used to unwind viral RNA during replication (Li et al., J. Virol. 73:3108-3116, 1999). While the exact functions of NS4A and NS4B remain to be elucidated, they are thought to be involved in RNA replication and immune modulation (Lindenbach and Rice, In: Fields Virology, Knipe and Howley, eds., Lippincott, Williams, and Wilkins, 991-1041, 2001; Muñoz-Jordán et al., Proc. Natl. Acad. Sci. USA 100:14333-14338, 2003; Muñoz-Jordán et al., J. Virol. 79:8004-8013, 2005). Finally, the NS5 protein is an RNA-dependent RNA polymerase involved in genome replication (Rice et al., Science 229:726-733, 1985). NS5 also shows methyltransferase activity commonly found in RNA capping enzymes and modulates the antiviral response through interferon antagonism (Koonin, J. Gen. Virol. 74:733-740, 1993; Best et al., J. Virol. 79:12828-12839, 2005).

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxyribonucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of nucleic acids. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Operably linked: A first nucleic acid is operably linked with a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. If introns are present, the operably linked DNA sequences may not be contiguous.

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, Philadelphia, Pa., 21^(st) Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more peptide conjugate, and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a carrier. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

Premembrane protein (prM): A flavivirus structural protein. The prM protein is an approximately 25 kDa protein that is the intracellular precursor for the membrane (M) protein. prM is believed to stabilize the E protein during transport of the immature virion to the cell surface. When the virus exits the infected cell, the prM protein is cleaved to the mature M protein, which is part of the viral envelope (Reviewed in Lindenbach and Rice, In: Fields Virology, Knipe and Howley, eds., Lippincott, Williams, and Wilkins, 991-1041, 2001).

Recombinant: A recombinant nucleic acid, protein, or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques such as those described in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule.

Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J. Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci., 85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins and Sharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res., 16:10881-90, 1988); Huang et al. (Comp. Appls. Biosci., 8:155-65, 1992); and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al. (Nature Genet., 6:119-29, 1994) presents a detailed consideration of sequence alignment methods and homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444-2448, 1988) may be used to perform sequence comparisons. ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the “Blast 2 sequences” function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The

BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-10, 1990; Gish and States, Nature Genet., 3:266-72, 1993; Madden et al., Meth. Enzymol., 266:131-41, 1996; Altschul et al., Nucleic Acids Res., 25:3389-402, 1997; and Zhang and Madden, Genome Res., 7:649-56, 1997.

Orthologs (equivalent to proteins of other species) of proteins are in some instances characterized by possession of greater than 75% sequence identity counted over the full-length alignment with the amino acid sequence of a specific protein using ALIGN set to default parameters. Proteins with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity.

When significantly less than the entire sequence is being compared for sequence identity, homologous sequences will typically possess at least 80% sequence identity over short windows of 10-20, and may possess sequence identities of at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or at least 99%, depending on their similarity to the reference sequence. Sequence identity over such short windows can be determined using LFASTA. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. Similar homology concepts apply for nucleic acids as are described for protein. An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that each encode substantially the same protein.

Structural protein: The capsid (C), premembrane (prM), and envelope (E) proteins of a flavivirus are the viral structural proteins. Flavivirus genomes consist of positive-sense RNAs that are roughly 11 kb in length. The genome has a 5′ cap, but lacks a 3′ polyadenylated tail (Wengler et al., Virology 89:423-437, 1978) and is translated into one polyprotein. The structural proteins (C, prM, and E) are at the amino-terminal end of the polyprotein followed by the non-structural proteins (NS1-5). The polyprotein is cleaved by virus and host derived proteases into individual proteins. The C protein forms the viral capsid while the prM and E proteins are embedded in the surrounding envelope. The E protein functions in binding to host cell receptors resulting in receptor-mediated endocytosis. In the low pH of the endosome, the E protein undergoes a conformational change causing fusion between the viral envelope and the endosomal membranes. The prM protein is believed to stabilize the E protein until the virus exits the infected cell, at which time prM is cleaved to the mature M protein (Reviewed in Lindenbach and Rice, In: Fields Virology, Knipe and Howley, eds., Lippincott, Williams, and Wilkins, 991-1041, 2001).

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals (such as mice, rats, sheep, horses, cows, and non-human primates).

Therapeutically effective amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a recombinant flavivirus useful for eliciting an immune response in a subject and/or for preventing infection by TBEV. Ideally, in the context of the present disclosure, a therapeutically effective amount of a recombinant flavivirus is an amount sufficient to increase resistance to, prevent, ameliorate, and/or treat infection caused by TBEV in a subject without causing a substantial cytotoxic effect in the subject. The effective amount of a recombinant flavivirus useful for increasing resistance to, preventing, ameliorating, and/or treating infection in a subject will be dependent on, for example, the subject being treated, the manner of administration of the therapeutic composition and other factors.

Tick-borne encephalitis virus (TBEV): A complex of related viruses that cause neurotropic disease. The TBEV complex includes Far Eastern, Siberian, and Central European TBEV subtypes, as well as Omsk hemorrhagic fever, Kyasanur forest disease, Langat, Louping ill, Negishi, and Powassan viruses. TBEV is transmitted through the bite of an infected ticks, mainly by Ixodes ricinus, I. persculatus ticks, or Dermacentor and Hyalomma species of ticks.

TBEV is endemic to many regions of the world (including Europe, Siberia, India, Japan, and North America); however the majority of cases occur in Russia. TBEV symptoms include a characteristic biphasic illness, with an initial phase of symptoms including fever, malaise, anorexia, muscle aches, headache, nausea and/or vomiting that lasts 2 to 4 days and corresponds to the viremic phase. After about 8 days of remission, the second phase of the disease occurs in 20 to 30% of patients and involves the central nervous system with symptoms of meningitis (e.g., fever, headache, and a stiff neck) or encephalitis (e.g., drowsiness, confusion, sensory disturbances, and/or motor abnormalities such as paralysis) or meningoencephalitis. In some patients infected with TBEV long-term or even permanent neurological sequelae occur.

Transformed: A “transformed” cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides, or nucleic acids derived from them. An attenuated vaccine is a virulent organism that has been modified to produce a less virulent form, but nevertheless retains the ability to elicit antibodies and cell-mediated immunity against the virulent form. A killed vaccine is a previously virulent microorganism that has been killed with chemicals or heat, but elicits antibodies against the virulent microorganism. Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.

III. TBEV/DEN4 Chimeric Viruses

Disclosed herein are chimeric TBEV/DEN4 flaviviruses including 5′ and 3′ non-coding regions, a C protein, and non-structural proteins from DEN4 virus, and a prM protein and E protein from TBEV. These chimeric viruses are designated TBEV/DEN4 chimeras. In particular examples, the chimeric TBEV/DEN4 virus includes a first nucleic acid molecule including a 5′ non-coding region from a DEN4 virus, a nucleic acid encoding a C protein from DEN4 virus, non-structural proteins from a DEN4 virus, and a 3′ non-coding region from a DEN4 virus, wherein nonstructural protein NS4B includes a phenylalanine at amino acid position 112, nonstructural protein NS5 includes an alanine at amino acid position 654 and an alanine at amino acid position 655, and the 3′ non-coding region includes a deletion of nucleotides 10478-10507 (430). The chimeric TBEV/DEN4 virus also includes a second nucleic acid molecule which is operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a prM protein from a TBEV and an E protein from a TBEV, wherein the E protein includes an amino acid substitution that differs from the wild type TBEV at amino acid position 315 and a tryptophan at amino acid position 240.

In some embodiments, the disclosed chimeric TBEV/DEN4 viruses are utilized in a replication-defective virus system or pseudoinfectious virus system. In these systems, the TBEV/DEN4 chimera includes one or more alterations that make the virus incapable of replicating its genome or incapable of assembling and releasing progeny virus particles. In particular examples, the chimeric TBEV/DEN4 viruses disclosed herein are provided in two separate components. When both components infect the same cell, genome replication or production of infectious progeny does not occur; however infectious progeny are limited following subsequent infection because one component is missing from the viral genome. However, after subsequent infection, viral gene expression occurs, producing viral antigens that can elicit an immunogenic response in a subject. See, e.g., U.S. Pat. Publication Nos. 2009/0155301 and 2009/0324623; Widman et al., Adv. Virus Res. 72:77-126, 2008; Widman et al., Vaccine 26:2762-2771, 2008; Ishikawa et al., Vaccine 26:2772-2781, 2008; Suzuki et al., J. Virol. 83:1870-1880, 2009, each of which is incorporated by reference herein.

In some embodiments, the disclosed chimeric TBEV/DEN4 viruses are modified to delete at least a portion of the nucleic acid encoding at least one structural protein, for example, the C protein, the prM protein and/or the E protein. In some examples a portion or all of the nucleic acid encoding the DEN4 C protein is removed from the chimeric virus. In other examples, the nucleic acids encoding the TBEV prM protein and/or E protein are removed from the chimeric virus. In a further example, the nucleic acids encoding the DEN4 C protein and the TBEV prM and E proteins are removed from the chimeric virus. In one non-limiting example the TBEV/DEN4 flavivirus includes 5′ and 3′ non-coding regions and non-structural proteins from DEN4 virus, and a prM protein and E protein from TBEV. In particular examples, the chimeric TBEV/DEN4 virus includes a first nucleic acid molecule including a 5′ non-coding region from a DEN4 virus, a nucleic acid encoding non-structural proteins from a DEN4 virus, and a 3′ non-coding region from a DEN4 virus, wherein nonstructural protein NS4B includes a phenylalanine at amino acid position 112, nonstructural protein NS5 includes an alanine at amino acid position 654 and an alanine at amino acid position 655, and the 3′ non-coding region includes a deletion of nucleotides 10478-10507 (Δ30) and also includes a second nucleic acid molecule which is operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a prM protein from a TBEV and an E protein from a TBEV, and wherein the E protein includes an amino acid substitution that differs from the wild type TBEV at amino acid position 315 and a tryptophan at amino acid position 240, wherein the chimeric virus does not encode a C protein. In one example, the chimeric TBEV/DEN4 flavivirus construct includes aspartic acid at amino acid 315 of the TBEV E protein. The nucleic acid encoding the at least one deleted structural protein is provided in a separate nucleic acid. For example a nucleic acid molecule including a nucleic acid encoding a DEN4 C protein, a TBEV prM protein and/or a TBEV E protein is provided in a separate construct.

In some examples disclosed herein, the TBEV prM and E protein-encoding nucleic acid is derived from a particular TBEV subtype, such as Far Eastern, Central European, or Siberian subtypes. In a particular example, the TBEV subtype includes Far Eastern subtype, Sofjin strain (e.g., GenBank Accession No. AB062064, incorporated by reference as present in GenBank on May 28, 2009). In other examples, the TBEV subtype includes European subtype, such as Hypr strain (e.g., GenBank Accession No. U39292) or Absettarov strain (e.g., GenBank Accession No. AF091005), both of which are incorporated by reference as in GenBank on May 28, 2009. In additional examples, the TBEV prM and/or E protein-encoding nucleic acid is derived from a member of the TBEV complex, such as Kyasanur forest disease, Langat, Louping ill, Negishi, Omsk hemorrhagic fever, or Powassan virus.

Nucleic acid and protein sequences for TBEV prM and E protein are publicly available. For example, GenBank Accession Nos.: NC_(—)001672 (nucleotides 469-972) and X03870 (nucleotides 339-890) disclose TBEV prM nucleic acid sequences, and GenBank Accession Nos.: NP_(—)775501, CAA27502, and X07755 (amino acids 466-963) disclose TBEV prM protein sequences, all of which are incorporated by reference as present in GenBank on May 28, 2009. In addition, GenBank Accession Nos.: NC_(—)001672 (nucleotides 973-2460) and X03870 (nucleotides 891-2100) disclose TBEV E nucleic acid sequences, and GenBank Accession Nos.: NP_(—)775503, CAA27504, and X07755 (amino acids 964-2457) disclose TBEV E protein sequences, all of which are incorporated by reference as present in GenBank on May 28, 2009. In certain examples, a TBEV prM and/or E sequence has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a publicly available TBEV prM and/or E sequence, such as those present in GenBank.

In further examples, the TBEV nucleic acid encoding the prM and/or E protein is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the prM or E protein-encoding nucleic acid sequence disclosed in SEQ ID NO: 1. In other examples, the TBEV prM and/or E proteins are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the prM or E protein amino acid sequence disclosed in SEQ ID NO: 2.

In some examples disclosed herein, the DEN4 5′ non-coding region, C protein, non-structural proteins, and 3′ non-coding region nucleic acid is derived from a particular DEN4 strain, such as wild type DEN4 1036 or attenuated DEN4 PDK-48. Additional DEN4 strains are known in the art (see e.g., U.S. Pat. Nos. 5,939,254 and 6,793,488). In a particular example, the DEN4 virus strain is DEN4 814669, Dominica 1981 (e.g., GenBank Accession Nos. AF375822 and AY376438, incorporated by reference as present in GenBank on May 28, 2009).

Nucleic acid and protein sequences for DEN4 viruses are publicly available. For example, GenBank Accession Nos.: NC_(—)002640 and AF326825 disclose DEN4 genomic nucleic acid sequences, and GenBank Accession Nos.: NP 073286 and AAG45435 disclose DEN4 protein sequences, all of which are incorporated by reference as present in GenBank on May 28, 2009. In certain examples, a DEN4 5′ non-coding region, C protein, non-structural proteins, and/or 3′ non-coding region sequence has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a publicly available DEN4 sequences, such as those present in GenBank.

In further examples, the DEN4 5′ non-coding region, C protein, non-structural proteins, and 3′ non-coding region nucleic acid is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the 5′ non-coding region, C protein, non-structural proteins, and 3′ non-coding region nucleic acid sequence disclosed in SEQ ID NO: 1. In other examples, the DEN4 C protein and non-structural proteins are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the C protein and non-structural protein amino acid sequence disclosed in SEQ ID NO: 2.

In particular embodiments, the chimeric TBEV/DEN4 virus includes amino acid substitutions at least at amino acid positions 240 and 315 of the TBEV E protein. An arginine residue at amino acid position 240 of the TBEV E protein is replaced with tryptophan. A lysine residue at amino acid position 315 of the TBEV E protein is replaced with a different amino acid (e.g., aspartic acid, alanine, phenylalanine, leucine, serine, arginine, threonine, tryptophan, valine, or tyrosine). In one example, the TBEV E protein includes tryptophan at amino acid 240 and aspartic acid at amino acid 315. The disclosed TBEV/DEN4 chimera may optionally include an amino acid substitution at position 84 of the TBEV E protein (such as glycine, leucine, valine, arginine, serine, alanine, tryptophan, or phenylalanine) In a particular example, the TBEV E protein includes a glycine at amino acid position 84.

In particular embodiments, the chimeric TBEV/DEN4 virus also includes amino acid substitutions at least at amino acid position 112 of the DEN4 NS4B protein, amino acid position 654 of the DEN4 NS5 protein, and amino acid position 655 of the DEN4 NS5 protein. A leucine residue at amino acid 112 of the DEN4 NS4B protein is replaced with phenylalanine, an aspartic acid at amino acid position 654 of the DEN4 NS5 protein is replaced with alanine, and an arginine at amino acid position 655 of the DEN4 NS5 protein is replaced with an alanine In addition, the TBEV/DEN4 chimera includes a deletion of 30 base pairs in the 3′ NCR at position 10478-10507 of the wild-type virus. The disclosed TBEV/DEN4 chimera may optionally include an amino acid substitution at position 6 of the DEN4 NS 1 protein (such as valine, cysteine, histidine, tryptophan, or tyrosine). In a particular example, the DEN4 NS 1 protein includes a valine at amino acid position 6. The TBEV/DEN4 chimera may optionally include an amino acid substitution at position 642, 643, 878, 879, or combinations thereof, of the DEN4 NS5 protein. In particular examples, one or more of amino acid positions 642, 643, 878, and/or 879 of the DEN4 NS5 protein includes an alanine.

In a particular embodiment, a chimeric TBEV/DEN4 virus has the nucleic acid or amino acid sequence disclosed herein as SEQ ID NOs: 1 and 2, respectively. In some examples, the chimeric TBEV/DEN4 virus encodes a polypeptide at least 95% (such as at least 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 2. In other examples, the chimeric TBEV/DEN4 virus encodes a polypeptide that comprises or consists of SEQ ID NO: 2. In further examples, the chimeric TBEV/DEN4 virus includes a nucleic acid molecule at least 95% (such as at least 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1. In other examples, the chimeric TBEV/DEN4 virus comprises or consists of SEQ ID NO: 1.

In one embodiment, a chimeric TBEV/DEN4 virus includes a nucleic acid encoding the virus that is represented by ATCC Accession number PTA-9968 (which is an E. coli strain BD1528 containing plasmid pBR322 including the chimeric TBEV/DEN4 genome TBE/DEN4Δ30/E-315D/NS5-654AA, provided to the ATCC on Apr. 17, 2009 and acknowledged as viable on May 28, 2009).

The disclosure also provides TBEV/DEN4 chimeras further including one or more nucleic acid or amino acid substitutions, such that the resulting chimera has improved characteristics. In some examples, the improved characteristic of the chimera with one or more substitutions includes, but is not limited to, decreased plaque size, temperature sensitivity, host range restriction, and increased stability in cell culture. In some examples, an improved characteristic includes reduced viral replication in neuronal cells. In additional examples, the improved characteristic of the chimera with one or more substitution includes decreased neurovirulence or neuroinvasiveness in a subject (such as mice or non-human primates).

Manipulation of the nucleic acid molecule of the disclosed TBEV/DEN4 chimeras (e.g. SEQ ID NO: 1) by standard procedures, including for instance site-directed mutagenesis or PCR and M13 primer mutagenesis, can be used to produce variants with improved characteristics (such as increased stability in cell culture, decreased neurovirulence or decreased neuroinvasiveness). Chemical mutagenesis may also be used to produce variants. Details of these techniques are well known. For instances, protocols are provided in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar physiochemical and/or structural properties. These so-called conservative substitutions are likely to have minimal impact on the activity and/or structure of the resultant protein. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Examples of conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

In addition to targeted mutagenesis to produce variants of the disclosed TBEV/DEN4 chimeras, naturally occurring variants may accrue upon passage in cell culture that result in variants, some with desirable characteristics. Nucleic acid and amino acid substitutions that accrue in chimeric viruses during cell culture passages are readily determined by sequence analysis of the virus amplified from isolated plaques of the virus seed, and can be engineered into infectious clones to generate TBEV/DEN4 chimera variants that have improved characteristics (such as decreased neurovirulence or neuroinvasiveness). Consistent variants identified from multiple seeds or isolated plaques indicate the desired substitutions of the chimera in the cell type.

In some embodiments, the TBEV/DEN4 chimera encodes a polypeptide that includes one or more amino acid substitutions of one or more residues of the TBEV prM or E protein, such that the chimera has improved characteristics. In other examples, the chimeric flavivirus encodes a polypeptide that includes one or more amino acid substitutions of one or more residues of the DEN4 non-structural proteins and/or C protein, such that the resulting chimera has improved characteristics. In additional examples, the chimeric flavivirus includes one or more nucleic acid substitutions in the DEN4 5′ and/or 3′ non-coding region, such that the chimera has improved characteristics.

The disclosed TBEV/DEN4 chimeras are produced by replication in host cells in culture. Methods of producing viruses are well known in the art (see e.g. Fields Virology, Knipe and Howley, eds., Lippincott, Williams, and Wilkins, 2001; Flint et al., Principles of Virology, ASM Press, 2000). Host cell lines should be easy to infect with virus or transfect with viral genomic RNA, be capable of stably maintaining foreign RNA with an unarranged sequence, and have the necessary cellular components for efficient transcription, translation, post-translation modification, virus assembly, and secretion of the protein or virus particle. Preferably, cells are those having simple media component requirements which can be adapted for growth in suspension culture. In some examples, the host cell line is a mammalian cell line that can be adapted to growth in low serum or serum-free medium. Suitable host cell lines include Vero (monkey), SH-SY5Y cells (human), and LN-18 cells (human), and C6/36 cells (mosquito). Suitable cell lines can be obtained from the American Type Culture Collection (ATCC), Manassas, Va.

The neurovirulence and neuroinvasiveness of the disclosed TBEV/DEN4 chimeric viruses can be assessed by methods well known to one of skill in the art. For example, neurovirulence may be assessed in animal models (such as mice or non-human primates) by ic inoculation with serial dilutions of virus and monitoring for morbidity and mortality. The ic 50% lethal dose (icLD₅₀) can be determined as a measure of neurovirulence. Neuroinvasiveness may be assessed by ip inoculation of individuals with virus and determining the ipLD₅₀ by monitoring animals for morbidity (such as neuroinflammation) and mortality, and measuring the presence of virus in the central nervous system at various time points post-inoculation.

IV. Methods of Eliciting an Immune Response

Provided herein are methods of eliciting an immune response in a subject by administering one or more of the disclosed nucleic acid chimeras, or a recombinant flavivirus comprising a nucleic acid chimera described herein, to the subject. In a particular example, the subject is a human. The recombinant flavivirus comprising a nucleic acid chimera disclosed herein can be used to produce an immune response that prevents infection with TBEV (such as Far Eastern, Central European, or Siberian TBEV subtypes), and can also be used to treat or inhibit infection with TBEV.

The E protein of members of the tick-borne encephalitis virus complex share significant sequence identity (approximately 85% or more). Thus, the TBEV/DEN4 chimeric viruses disclosed herein may also be used to produce an immune response that prevents or treats or inhibits infection with other members of the tick-borne encephalitis virus complex, including, but not limited to Omsk hemorrhagic fever, Kyasanur forest disease, Langat, Louping ill, Negishi, and Powassan viruses.

In some examples, the method further includes selecting a subject in need of enhanced immunity to TBEV (such as Far Eastern, Central European, or Siberian TBEV subtypes). Subjects in need of enhanced immunity to TBEV include subjects who are at risk of TBEV infection, subjects who have been exposed to one or more TBEV, and subjects who are infected with TBEV. Residents of, or travelers to, countries or regions where TBEV is endemic are at risk of contracting TBEV, such as TBEV caused by infection with Far Eastern, Central European, or Siberian TBEV subtypes. Additional factors that contribute to risk of infection with TBEV include the characteristics of the area, presence of TBEV in the area, exposure to ticks, and lack of preventive measures (such as insect repellant). The risk of TBEV is generally higher in rural areas and among those with recreational or occupational exposure to outdoor settings (such as farm or forest workers, hunters, and campers).

A relatively recent development in the field of immune stimulatory compounds (for example, vaccines) is the direct injection of nucleic acid molecules encoding peptide antigens (broadly described in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med.

334:42-45, 1996). Vectors that include nucleic acid molecules described herein, or that include a nucleic acid encoding a virus polypeptide comprising at least one virus epitope may be utilized in such DNA vaccination methods.

One approach to administration of nucleic acids is direct immunization with vector DNA. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Pat. No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Pat. Nos. 5,593,972 and 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Pat. No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune-stimulating constructs, or ISCOMS™, which are negatively charged cage-like structures of 30-40 nm in size formed spontaneously on mixing cholesterol and saponin (see, e.g., Sjolander et al., J. Leukoc. Biol. 64:713-723, 1998). Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™ have been found to produce Class I mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

The disclosed nucleic acid chimeras or recombinant flaviviruses comprising the nucleic acid chimeras can be administered to a subject by any of the routes normally used for introducing a composition into a subject. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Administration can be systemic or local.

Immunogenic compositions are administered in any suitable manner, such as with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Administration can be accomplished by single or multiple doses. The dose administered to a subject in the context of the present disclosure should be sufficient to induce a beneficial therapeutic response in a subject over time, or to inhibit or prevent infection. The dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the particular immunogenic composition being used, and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art using only routine experimentation. In some examples, the dose administered to the subject is about 1 μg to about 1000 μg (such as about 1 μg to about 500 μg, about 10 μto about 500 μg, or about 50 μg to about 500 μg) of the disclosed chimeric viruses. In other examples, the dose administered to the subject is about 10 pfu to about 10⁵ pfu (such as about 10 pfu to about 10⁴ pfu, about 10² pfu to about 10⁴ pfu, or about 10³ pfu to about 10⁵ pfu). Repeated immunizations may be necessary to produce an immune response in a subject. Immunization protocols (such as amount of immunogen, number of doses and timing of administration) can be determined experimentally, for example by using animal models (such as mice or non-human primates), followed by clinical testing in humans.

In some examples, the disclosed TBEV/DEN4 chimeric nucleic acid or virus is administered to a subject as a two-component genome or as a pseudoinfectious virus. See, e.g., U.S. Pat. Publication Nos. 2009/0155301 and 2009/0324623; Widman et al., Adv. Virus Res. 72:77-126, 2008; Widman et al., Vaccine 26:2762-2771, 2008; Ishikawa et al., Vaccine 26:2772-2781, 2008; Suzuki et al., J. Virol. 83:1870-1880, 2009, each of which is incorporated by reference herein.

In one example, the method includes administering a therapeutically effective amount of a pseudoinfectious TBEV/DEN4 chimeric virus to a subject. The pseudoinfectious TBEV/DEN4 can be produced by transfecting cells with a disclosed chimeric TBEV/DEN4 virus modified to delete at least a portion of the nucleic acid encoding at least one structural protein (for example, the C protein, prM protein and/or E protein), and also transfecting the same cells with a second viral genome capable of producing the deleted structural proteins (such as the C protein, prM protein and/or E protein). The resulting infectious particles can be administered to a subject to elicit an immune response to expressed viral proteins, such as the prM and E proteins.

In another example, the method includes administering a therapeutically effective amount of a two-component TBEV/DEN4 genome including a TBEV/DEN4 viral genome including a disclosed chimeric TBEV/DEN4 virus with deletion of the C protein, prM protein and/or E protein, and a complementing genome that includes a nucleic acid encoding the deleted structural protein (such as a DEN4 C protein, TBEV prM protein and/or TBEV E protein).

In additional examples, the disclosed nucleic acid chimeras, recombinant flaviviruses comprising the nucleic acid chimeras, or immunogenic compositions can be administered to a subject prior to, concurrent with or subsequent to one or more additional immunogenic compositions, such as one or more vaccines for TBEV, other flaviviruses (such as Dengue viruses, St. Louis encephalitis virus, Japanese encephalitis virus, West Nile virus, or yellow fever virus), or other pathogens. In a particular example, the additional immunogenic composition includes a Langat virus/DEN4 chimera (see, e.g., Pletnev et al., J. Virol. 75:8259-8267, 2001). In another particular example, the additional immunogenic composition includes a West Nile virus/DEN4 chimera (see, e.g., Pletnev et al., Proc. Natl. Acad. Sci. USA 99:3036-3041, 2002).

Provided herein are pharmaceutical compositions (also referred to as immunogenic compositions) which include a therapeutically effective amount of the disclosed recombinant TBEV/DEN4 viruses alone or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil.

The immunogenic compositions disclosed herein can additionally employ adjuvants conventionally found in pharmaceutical compositions in their art-established fashion and at their art-established levels. Thus, for example, the compositions can contain materials useful in physically formulating various dosage forms of the preferred embodiments. Adjuvants are commonly combined with vaccines for the purpose of improving immune response. Suitable adjuvants include aluminum hydroxide, aluminum phosphate, aluminum oxide, monophosphenyl lipid A, muramyl dipeptide, glucans, Quil A, Freund's incomplete adjuvant, or Freund's complete adjuvant.

V. Methods of Assessing Immunogenic Response

The compositions and methods disclosed herein are useful for generating an immunogenic (immunological) response in a host or subject. Methods of assessing an immune response to a composition are well known to one of skill in the art.

In some examples, an immune response to a recombinant TBEV/DEN4 chimeric virus, such as one of the disclosed chimeras, is determined by assessing the production of antibodies to the specific viral proteins included in the chimeric virus (such as TBEV prM and E proteins). The method of detecting antibodies to the disclosed recombinant TBEV/DEN4 chimeric viruses in a sample can be performed, for example, by a plaque reduction neutralization test. In some examples, TBEV/DEN4 virus is incubated with serial dilutions of fluid sample specimens. The neutralizing antibody titer is identified as the highest serum dilution that reduces the number of virus plaques in the test by 60% or greater. A fluid sample of this method can comprise any biological fluid which could contain the antibody, such as cerebrospinal fluid, blood, bile plasma, serum, saliva, and urine. Other possible examples of body fluids include sputum, mucus and the like. The durability of the neutralizing antibody response can be assessed by determining neutralizing antibody titer at various time points post-inoculation (such as about one month, two months, three months, six months, nine months, twelve months, or more post-inoculation).

Enzyme immunoassays such as IFA, ELISA and immunoblotting can be readily adapted to accomplish the detection of antibodies according to the methods of this disclosure. An ELISA method effective for the detection of the antibodies can, for example, be as follows: 1) bind a polypeptide (such as TBEV prM or E protein) to a substrate; 2) contact the bound polypeptide with a fluid or tissue sample containing the antibody; 3) contact the above with a secondary antibody bound to a detectable moiety which is reactive with the bound antibody (for example, horseradish peroxidase enzyme or alkaline phosphatase enzyme); 4) contact the above with the substrate for the enzyme; 5) contact the above with a color reagent; and 6) observe/measure color change or development.

Another immunologic technique that can be useful in the detection of antibodies uses monoclonal antibodies (mAbs) for detection of antibodies specifically reactive with the disclosed TBEV/DEN4 chimera (such as TBEV prM or E protein) in a competitive inhibition assay. Briefly, a sample is contacted with a polypeptide which is bound to a substrate (for example, a 96-well plate). Excess sample is thoroughly washed away. A labeled (for example, enzyme-linked, fluorescent, radioactive, etc.) mAb is then contacted with any previously formed polypeptide-antibody complexes and the amount of mAb binding is measured. The amount of inhibition of mAb binding is measured relative to a control (no antibody), allowing for detection and measurement of antibody in the sample.

As a further example, a micro-agglutination test can be used to detect the presence of antibodies to the disclosed TBEV/DEN4 chimeras in a sample. Briefly, latex beads, red blood cells or other agglutinable particles are coated with a polypeptide of the TBEV/DEN4 chimera (such as TBEV prM or E protein) and mixed with a sample, such that antibodies in the sample which are specifically reactive with the antigen crosslink with the antigen, causing agglutination. The agglutinated polypeptide-antibody complexes form a precipitate, visible with the naked eye or measurable by spectrophotometer.

In yet another example, a microsphere-based immunoassay can be used to detect the presence of antibodies in a sample. Briefly, microsphere beads are coated with a component of the disclosed TBEV/DEN4 chimeras (such as TBEV prM or E protein) and mixed with a sample, such that antibodies in the sample which are specifically reactive with the antigen bind the antigen. The bead-bound polypeptide-antibody complexes are allowed to react with fluorescent-dye labeled anti-species antibody (such as FITC-labeled goat anti-human IgM), and are measured using a microsphere reader (such as a Luminex instrument).

In additional examples, an immune response to a TBEV/DEN4 chimera (such as TBEV prM or E protein) is determined by assessing the protective effect against infection produced by immunization with the disclosed TBEV/DEN4 chimeras. Briefly, a host (such as a mouse or a non-human primate, for example rhesus monkey) is immunized with one or more of the disclosed TBEV/DEN4 chimeric viruses. Following a sufficient period of time to allow development of an immune response, the host is challenged with TBEV or unmodified TBEV/DEN4 chimera. The infection is monitored by examination of blood samples for the presence of virus in the blood. A reduction in the viral titer in immunized hosts as compared to control hosts and/or an increase in TBEV-specific neutralizing antibody titers indicates that an immune response developed to the composition. Protective immunity is assessed by absence of viremia, absence of clinical symptoms of infection, and measurement of host anamnestic response following challenge with TBEV or unmodified TBEV/DEN4 chimera.

The efficacy of the disclosed TBEV/DEN4 chimeras to protect against infection with other members of the TBEV complex (such as Omsk hemorrhagic fever, Kyasanur forest disease, Langat, Louping ill, Negishi, and Powassan virus) can be similarly assessed, except the challenge is with the appropriate TBEV complex virus, rather than TBEV or unmodified TBEV/DEN4 virus.

The present disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Construction of TBEV/DEN4 Chimeras and Characterization in Cell Culture

This example describes the construction of TBEV/DEN4 nucleic acid chimeras and their in vitro characterization.

Methods

Cell culture and viruses. Simian Vero cells (World Health Organization seed, passages 143-149) were maintained in Opti-Pro Serum Free Medium (Invitrogen, Carlsbad, Calif.), supplemented with 4 mM L-glutamine (Invitrogen). Human neuroblastoma SH-SY5Y cells (American Type Culture Collection, Manassas, Va.) were maintained in 1:1 Minimal Essential and F12 media (Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Basel, Switzerland). Human glioblastoma LN-18 cells (American Type Culture Collection, Manassas, Va.) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen), supplemented with 5% heat-inactivated FBS, 4 mM L-glutamine, and 1.5 g/L sodium bicarbonate (Invitrogen).

Construction of full-length cDNA clones and recovery of chimeric viruses. Chimeric TBEV/DEN4 virus contained the prM and E protein genes of TBEV Far-Eastern subtype strain Sofjin and the remaining sequence derived from recombinant DEN4 virus. Chimeric TBEV/DEN4Δ30 virus also contained a 30 nucleotide deletion (nucleotides 10478-10507) within the 3′ non-coding region of the DEN4 genome. Construction of both viruses has been described previously (Pletnev et al., Proc. Natl. Acad. Sci. USA 89:10532-10536, 1992; Rumyantsev et al., Vaccine 24:133-143, 2006). The full-length infectious cDNA clones TBEV/DEN4 and TBEV/DEN4Δ30 (GenBank Accession Nos. FJ828986 and FJ828987, respectively; incorporated herein by reference on May 28, 2009) were used in these studies to generate recombinant viruses containing amino acid substitutions of aspartic acid at amino acid position 315 of the TBEV E protein (E-K₃₁₅D) and alanine at amino acid positions 654 and 655 of the DEN4 NS5 protein (NS5-DR_(654,655)AA). Each amino acid substitution was introduced singly or in combination by site-directed mutagenesis.

DNA fragments encompassing either DEN4 or TBEV specific sequences were sub-cloned into the pUC18 vector and each amino acid substitution was introduced through site-directed mutagenesis of the plasmid, as previously described (Rumyantsev et al., J. Virol. 80:1427-1439, 2006; Hanley et al., J. Virol. 76:525-531, 2002). Mutagenic primers introducing Asp (codon GAC) at amino acid residue 315 (nucleotides 1893 and 1895) of the TBEV E protein in pUC-TBEV and Ala/Ala (codons GCA & GCG) at amino acid residues 654 and 655 (nucleotides 9538, 9539, 9540, 9541) of the DEN4 NS5 protein in pUC-DEN4c were used to engineer these mutations. The pUC18-TBEV fragment contained unique NheI and XhoI restriction sites that corresponded to TBEV/DEN4 nucleotides 240-2361, while the pUC-DEN4c fragment contained unique SacII and MluI sites that corresponded to TBEV/DEN4 nucleotides 9334-10418. Fragments containing the desired mutations were excised from pUC-TBEV or pUC-DEN4c by restriction digestion and introduced into the TBEV/DEN4 or TBEV/DEN4Δ30 infectious clones containing an SP6 promoter (Lai et al., Proc. Natl. Acad. Sci. USA 88:5139-5143, 1991; Pletnev et al., Proc. Natl. Acad. Sci. USA 89:10532-10536, 1992; Rumyantsev et al., Vaccine 24:133-143, 2006).

Full-length infectious chimeric RNA derived from the modified TBEV/DEN4 or TBEV/DEN4Δ30 DNA plasmids were generated by transcription with SP6 polymerase (EpiCentre Biotechnologies, Madison, Wis.) and transfected into Vero cells using Lipofectamine™ (Invitrogen, Carlsbad, Calif.), according to the manufacturer's protocol. Since mutations at these positions previously resulted in temperature sensitivity in either LGTV or DEN4V, all viruses were grown at 32° C. The rescued TBEV/DEN4 and TBEV/DEN4Δ30 mutant viruses were biologically cloned by two terminal dilutions and then amplified by two passages in Vero cells before experimental stocks were obtained. Titers for all viruses obtained were between 6.4 and 7.2 log₁₀ pfu/ml.

Titration of chimeric viruses. Confluent monolayers of Vero cells in 24-well plates were infected with 10-fold serial dilutions of virus, incubated at 37° C. for one hour, and then overlaid with 1 ml of Opti-MEM I containing 1% methylcellulose (Invitrogen), 2% heat-inactivated FBS, 4 mM L-glutamine, and 0.05 mg/ml of gentamicin. After incubation for 6 days at 32° C., the cells were fixed in 100% methanol for 20 minutes, and plaques were visualized by immunostaining with TBEV-specific hyperimmune mouse ascitic fluid (ATCC, Catalog No. VR-1264 AF) and peroxidase-labeled polymer conjugated to anti-mouse immunoglobulin (Dako Co., Carpinteria, Calif.)

Sequencing of cDNA clones and chimeric viruses. Viral RNA was extracted from virus suspension using the QiaAmp® Viral RNA mini kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocol. One-step RT-PCR was performed on the viral RNA using Superscript® One-Step kit (Invitrogen), and primers specific for the DEN4 virus or TBEV genome (Table 2). The nucleotide consensus sequences were determined through direct sequence analysis of the PCR fragments on a 3730 Genetic Analyzer using TBEV- or DEN4-specific primers in a BigDye® cycle sequencing reaction (Applied Biosystems, Foster City, Calif.) and were analyzed using Sequencher® 4.7 software (Gene Codes Corporation, Ann Arbor, Mich.) as described previously (Rumyantsev et al., Vaccine 24:133-143, 2006).

TABLE 2 Primers used to derive cDNA amplicons Nucleotide position in TBEV/DEN4 genome Primer (SEQ ID name Primer sequence NO: 1) 5′DEN4 AGTTGTTAGTCTGTGTGGACCGACA  1-24 (SEQ ID NO: 3) 2546R TCTCGCTGGGGACTCTGGTTGAAAT 2536-2560 (SEQ ID NO: 4) 2420F AGCAGACATGGGTTGTGTGGCGTC 2434-2457 (SEQ ID NO: 5) 4588R ACTCCTTCAGACAGTGCGGCTTTT 4579-4602 (SEQ ID NO: 6) 4420F CCCTTTTGGTGAAACTGGCACTGA 4434-4457 (SEQ ID NO: 7) 6776R TTGATTGTCTTGTGGGGTCCTTTG 6767-6790 (SEQ ID NO: 8) 6497F CTATCAACACGCCCTGAACGAACT 6511-6534 (SEQ ID NO: 9) 8989R TACCAGATTGCTCGGCTTCCCTTG 8980-9003 (SEQ ID NO: 10) 8520F ATGGTGAACGGGGTGGTAAAACTG 8534-8557 (SEQ ID NO: 11) 10610R GCTCTGTGCCTGGATTGATGTTG 10571-10593 (SEQ ID NO: 12)

In vitro characterization of mutant viruses in cell culture. All mutant viruses were evaluated in a comparative study for temperature sensitivity (ts), host range restriction (hs) and small plaque (sp) phenotypes by assessing virus titers at 32° C., 35° C., 37° C., and 39° C. in simian kidney Vero, human neuroblastoma SHSY-5Y, or human glioblastoma LN-18 cells. The efficiency of plaque (EOP) formation was determined by infecting confluent monolayers of Vero, LN-18, or SHSY-5Y cells with 10-fold serially diluted virus for 1 hr at 37° C., after which Opti-MEM I overlay containing methylcellulose, FBS, and gentamicin was added to each well. Cells were incubated for 6 days at the indicated temperature and plaques were visualized by immunostaining, as described above. Mutant viruses that exhibited 100-fold or greater reduction in titer at a given temperature relative to its titer at 32° C. were considered to be ts, while viruses that demonstrated 100-fold or greater reduction of titer in neuronal cells at 32° C. compared to that in Vero cells were designated as having a hr phenotype. Mutant viruses with mean plaque diameters that were ≦50% of the size of the parental TBEV/DEN4 or TBEV/DEN4Δ30 virus on the given cell line were designated as being sp. The EOP assays were undertaken on three separate occasions and the mean data point for each sample was measured.

Results

Derivation of attenuated TBE/DEN4 and TBEV/DEN4Δ30 chimeric viruses. Full-length chimeric viruses containing the structural prM and E protein genes of the TBEV Far Eastern strain Sofjin and the remaining protein genes and NCRs of DEN4 virus, with or without the Δ30 mutation in the 3′ NCR were derived as previously described (Pletnev et al., Proc. Natl. Acad. Sci. USA 89:10532-10536, 1992; Pletnev et al., J. Virol. 67:4956-4963, 1993; Rumyantsev et al., Vaccine 24:133-143, 2006). Additional substitutions within the E glycoprotein and NS5 protein genes (E-K₃₁₅D and NS5-DR_(654,655)AA) were introduced, singly or in combination, into either the TBEV/DEN4 or TBEV/DEN4Δ30 infectious cDNA clones. Chimeric viruses containing these substitutions were generated and recovered from Vero cells. All terminally diluted viruses were sequenced throughout the structural and NS5-3′ NCR regions following expansion in Vero cells and were found to contain the desired mutations. All viruses maintained at least 6.0 log₁₀ pfu/ml titers at 32° C. in Vero cells (FIG. 1).

Full-length virus was also sequenced following expansion in Vero cells and found to include amino acid substitutions at position 240 of the TBEV E protein (E-R240W) and at position 112 of the DEN4 NS4B protein (NS4B-L112F). The nucleotide and amino acid sequence of the full-length TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimera, including the mutations arising in Vero cells is included herein as SEQ ID NOs: 1 and 2, respectively.

In vitro characterization of chimeric viruses. Host range restriction (hr) and temperature sensitivities (ts) of all chimeric TBEV/DEN4 or TBEV/DEN4Δ30 viruses were measured by infecting Vero or human neuronal cells (SH-SY5Y cells and LN-18 cells) at various temperatures and investigating viral titers and plaque morphology. All mutant viruses utilizing the unmodified TBEV/DEN4 backbone replicated well in all cell lines and at all temperatures, with the exception of TBEV/DEN4/E-K₃₁₅D/NS5-DR_(654,655)AA. This virus was ts in Vero, LN-18, and SHSY-5Y cells at 37° C. and 39° C., and replicated approximately 320-fold to 125,000-fold less at these temperatures than at 32° C.

TBEV/DEN4Δ30 virus was not hr or ts, as it replicated well up to 39° C. in all three cell lines investigated (FIG. 1). All viruses with the TBEV/DEN4Δ30 backbone demonstrated similar replication phenotypes whether they were grown in Vero or SH-SY5Y cells (FIG. 1). TBEV/DEN4Δ30 and TBEV/DEN4Δ30/E-K₃₁₅D viruses demonstrated stable replication phenotypes in both cell lines and replicated well up to 39° C. (≧5.7 log₁₀ pfu/ml), while TBEV/DEN4Δ30/NS5-DR_(654,655)AA demonstrated a 40,000-fold reduction in replication up to 39° C. in either cell line (FIG. 1). The triple mutant virus, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA, was the most attenuated and demonstrated a 400,000-fold reduction in replication at 39° C. At 35° C., all viruses still replicated well in these cell lines; however, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA replicated less well at 37° C. compared to the other three viruses in both cell lines, and was ts at the higher temperatures (FIG. 1).

While all viruses replicated well (between 6.4 and 7.8 log₁₀ pfu/ml) at 32° C. in Vero and SH-SY5Y cells, only TBEV/DEN4Δ30 and TBEV/DEN4Δ30/NS5-DR_(654,655)AA viruses replicated to similar titers in human glioblastoma LN-18 cells at 32° C. (FIG. 1). TBEV/DEN4Δ30/E-K₃₁₅D virus demonstrated a borderline hr phenotype in LN-18 cells compared to Vero cells at 32° C.; however, it was not ts up to 39° C. in any of the cell lines. TBEV/DEN4Δ30/NS5-DR_(654,655)AA virus was not hr, although it was ts in LN-18 cells at 37° C. and 39° C., as shown by a 320-fold to 160,000-fold reduction in replication at these temperatures (FIG. 1). The triple mutant virus, (TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA) was both ts and hr, as it demonstrated 100-fold to >400,000-fold reduction in titer at 37° C. and 39° C. compared to the permissive temperature in all three cell lines measured and approximately a 400-fold reduction of titer in LN-18 cells compared to its titer in Vero cells at the same temperature (4.0 log₁₀ vs. 6.6 log₁₀ pfu) (FIG. 1). Furthermore, the triple mutant virus always exhibited 50-75% smaller-sized plaques compared to the TBEV/DEN4Δ30 parental virus, regardless of which cell line or temperature was analyzed (Table 3). Therefore, while E-K₃₁₅D and NS5-DR_(654,655)AA mutations exerted various attenuating effects on the property of the virus, the hr, ts, and small plaque phenotypes of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA virus indicated that it was more attenuated in vitro compared to its parental or individually mutated viruses.

TABLE 3 Efficiency of plaquing of TBEV/DEN4Δ30 Chimeras 32° C. 35° C. 37° C. 39° C. Plaque Plaque Plaque Plaque Titer^(a) size^(b) Titer size Titer size Titer size Vero Cells TBEV/DENΔ30 7.8 1.0 7.5 1.0 7.1 1.0 6.0 0.5 TBEV/DENΔ30/E- 6.7 0.5 6.7 0.5 6.3 0.5 5.7 0.5 K₃₁₅D TBEV/DENΔ30/NS5- 7.4 0.5 6.9 0.3 4.0 ~0.2 2.8 ~0.2 DR_(654, 655)AA TBEV/DENΔ30/E- 6.6 ~0.3 5.8 ~0.2 3.0 <0.2 <1.0 ND K₃₁₅D/NS5- DR_(654, 655)AA LN18 Cells TBEV/DENΔ30 6.9 ~0.3 6.7 1.0 6.3 1.0 5.0 ~0.2 TBEV/DENΔ30/E- 4.8 ~0.2 5.3 0.5 4.9 0.5 3.0 <0.2 K₃₁₅D TBEV/DENΔ30/NS5- 6.5 <0.2 6.1 ~0.3 4.0 <0.2 1.3 <0.2 DR_(654, 655)AA TBEV/DENΔ30/E- 4.0 <0.1 3.0 <0.1 2.0 <0.1 <1.0 ND K₃₁₅D/NS5- DR_(654, 655)AA SH-SY5Y Cells TBEV/DENΔ30 7.8 2.0 7.9 3.0 7.3 3.0 6.5 1.0 TBEV/DENΔ30/E- 6.8 1.5 6.5 2.0 6.4 3.0 5.8 2.0 K₃₁₅D TBEV/DENΔ30/NS5- 7.2 1.5 6.7 2.0 6.7 1.0 2.6 1.0 DR_(654, 655)AA TBEV/DENΔ30/E- 6.4 1.0 6.0 1.0 3.7 ~0.3 <1.0 ND K₃₁₅D/NS5- DR_(654, 655)AA ^(a)Titer is calculated as log₁₀ pfu/ml ^(b)Plaque size calculated in mm ND, not determined. Bold font indicates 100-fold or greater reduction in viral replication compared to the permissive temperature (32° C.) in that particular cell line.

Example 2 Neurovirulence and Neuroinvasiveness of Chimeric Viruses in Mice

This example describes the characterization of the neurovirulence and neuroinvasiveness of the TBEV/DEN4 chimeras in mice.

Methods

To determine the neurovirulence of the TBEV/DEN4 chimeric viruses, litters of approximately ten 3 day-old Swiss mice (Taconic Farms, Hudson, N.Y.) were inoculated with 10-fold serial dilutions of virus via the intracerebral (ic) route and monitored for morbidity and mortality up to 21 days post-inoculation (dpi). Moribund mice were euthanized by CO₂. The ic 50% lethal dose (icLD₅₀) was determined by the method of Reed and Muench (Am. J. Hyg. 27:493-497, 1938).

Further studies were undertaken in litters of 3 day-old or 5 day-old suckling Swiss mice to investigate the replication of the chimeric viruses in mouse brain. The mice were ic inoculated with 10³ pfu of virus and at least three mouse brains per group were harvested on odd dpi, up to 21 dpi. Mouse brains were individually homogenized as a 10% solution (w/v) using homogenization buffer (1X Hank's Balanced Salt Solution (Invitrogen), 40 mg/ml ciprofloxacin (Bayer), 150 mg/ml clindamycin (Pharmacia & Upjohn), 250 μg/ml amphotericin B (Quality Biologicals)), as previously described (Blaney et al., Vaccine, 26:4150-4159, 2008). Clarified viral supernatants were titrated for viral loads in Vero cells. To investigate the stability of the engineered mutations within the chimeric viruses after their replication in brain, virus RNA was extracted from brain homogenates obtained on the last days they were positive for virus and consensus sequences of the genomic regions encompassing the engineered mutations from each group were directly determined.

To investigate virus-induced pathology of the viruses in brains, 3-week-old female C57BL/6 mice (Taconic Farms) in groups of three were inoculated is with 10⁴ pfu of either TBEV/DEN4, TBEV/DEN4Δ30, or TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA, whereas three control mice were mock-inoculated with Leibovitz's L-15 medium (Invitrogen). All mice were observed daily and euthanized on day 6, when TBEV/DEN4-infected mice developed paralysis. Mice were euthanized and perfused transcardially with PBS, and each mouse brain was dissected sagitally. The left hemisphere was frozen and stored at −80° C. for virus quantitation. The right hemisphere was fixed in 4% paraformaldehyde for 72 hours and processed according to standard histological methods. Twenty-five sections (30 μm thick) from each hemisphere were stained with hematoxylin and eosin (H&E), and analyzed for the presence and severity of virus-induced histopathology.

The neuroinvasive phenotype of the chimeric viruses was investigated in 3-week old SCID (ICRSC-M) mice (Taconic Farms, Hudson, N.Y.). To measure neuroinvasiveness, 10 mice were inoculated ip with 10⁵ pfu of TBEV/DEN4 virus, while separate groups of 33 to 56 mice were inoculated ip with 10⁵ pfu of TBEV/DEN4Δ30 virus or its derivatives. Mice were observed for 49 days for signs of morbidity typical of CNS involvement, including paralysis. Moribund mice were humanely euthanized upon signs neurologic disease. Kaplan-Meier survival curves followed by Tukey post-hoc tests were performed for statistical analysis (p<0.05) (GraphPad Prism 5 software, La Jolla, Calif.). Separately, groups of 35 SCID mice were inoculated ip with 10⁵ pfu of TBEV/DEN4 or TBEV/DEN4Δ30 virus, and the brains of three mice per group were harvested on odd days, for 21 days, to assess the level of virus replication. In addition, SCID mice in groups of 12 were inoculated ip with 10⁵ pfu of TBEV/DEN4Δ30-derived mutant viruses. The brains of three mice from each of these groups were harvested on days 13, 15, 17, and 19 to assess the level of virus replication as described above.

Results

Intracerebral LD₅₀ (icLD₅₀) values of TBEV/DEN4 or TBEV/DEN4Δ30 mutant viruses were measured in 3-day old Swiss mice to assess neurovirulence attenuation phenotypes of the mutant chimeric viruses in vivo. Introduction of the Δ30 deletion into the TBEV/DEN4 backbone did not alter the ability of the virus to infect the CNS, as icLD₅₀ values and average survival times (ASTs) were no different between TBEV/DEN4 and TBEV/DEN4Δ30 viruses in 3 day-old mice (Table 4). However, mutation of the E-K₃₁₅D or NS5-DR_(654,655)AA residues within the viral backbone alone decreased the overall mouse neurovirulence by 8-fold and 20-fold, respectively, and introduction of both substitutions concurrently into the parental backbone reduced mouse neurovirulence up to 50-fold (Table 4). Furthermore, the ASTs of these mice increased incrementally by the addition of E-K₃₁₅D, NS5-DR_(654,655)AA, and E-K₃₁₅D/NS5-DR_(654,655)AA, from 8.6 dpi to >21 dpi. Decreases in neurovirulence were also noted in TBEV/DEN4Δ30 viruses containing the single substitutions at E-K₃₁₅D or NS5-DR_(654,655)AA, or both substitutions as demonstrated by 4-fold to 487-fold increases in icLD₅₀ values. Although no difference in icLD₅₀ value was noted between TBEV/DEN4 and TBEV/DEN4Δ30 viruses, mice inoculated with TBEV/DEN4Δ30/E-K₃₁₅D or TBEV/DEN4Δ30/NS5-DR_(654,655)AA succumbed to infection approximately two to four days later than mice inoculated with TBEV/DEN4/E-K₃₁₅D or _(TBEV/DEN)4/NS5-DR_(654,655)AA (Table 4).

TABLE 4 Neurovirulence phenotypes of chimeric viruses in suckling Swiss mice Fold reduction in neurovirulence compared to Virus LD₅₀ (pfu) parental^(a) AST (days)^(b) TBEV/DEN4  0.8 —    7.4 TBEV/DEN4/E-K₃₁₅D  6.6  8    8.6 TBEV/DEN4/NS5-DR_(654,655)AA  16.2  20   12.2 TBEV/DEN4/E-K₃₁₅D/NS5-  40.8  51 >21   DR_(654,655)AA TBEV/DEN4Δ30  1.0 —    7.0 TBEV/DEN4Δ30/E-K₃₁₅D  4.1  4   10.3 TBEV/DEN4Δ30/NS5-  40.7  41  16  DR_(654,655)AA TBEV/DEN4Δ30/E- 487   487 >21   K₃₁₅D/NS5-DR_(654,655)AA ^(a)Parental virus is TBEV/DEN4 for the first set of chimeras or TBEV/DEN4Δ30 for the second set of chimeras. ^(b)Average survival times of mice that died after ic inoculation of 10 pfu of indicated virus.

Further analysis of neurovirulence in these mice revealed that introduction of the substitutions at NS5-DR_(654,655)AA attenuated the virus for mouse neurovirulence to a greater extent than either the substitution at E-K₃₁₅D or Δ30 alone. While icLD₅₀ values of E-K₃₁₅D increased between 4-fold and 8-fold compared to the parental viruses, introduction of NS5-DR_(654,655)AA into either the TBEV/DEN4 or TBEV/DEN4Δ30 backbone resulted in a decrease of mouse neurovirulence by 20-fold and 41-fold, respectively (Table 4). In addition, introduction of both Δ30 and NS5-DR_(654,655)AA into the TBEV/DEN4 backbone decreased the mouse neurovirulence phenotype compared to TBEV/DEN4/NS5-DR_(654,655)AA virus alone by 2.5-fold (40.7 vs. 16.2 pfu) and increased the AST by 4.5 days. Addition of both sets of amino acid substitutions led to an increase in AST by at least 3-fold compared to their parental viruses. While the Δ30 mutation did not alter the mouse neurovirulence phenotype by itself, it acted synergistically with the substitutions at E-K₃₁₅D and NS5-DR_(654,655)AA residues to increase the icLD₅₀ from 40.8 pfu to 487 pfu, and subsequently decrease mouse neurovirulence by 12-fold (Table 4).

The TBEV/DEN4Δ30 mutant viruses were also assayed for replication in 3- or 5-day-old suckling mouse brains (FIG. 2). Parental unmodified TBEV/DEN4Δ30 virus rapidly reached high viral titers in both sets of mice (between 7.6 and 8.7 log₁₀ PFU) by 5 dpi after ic inoculation. After ic inoculation of 3-day-old mice, TBEV/DEN4Δ30/NS5-DR_(654,655)AA and TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA chimeric viruses replicated approximately 100-fold less than either the TBEV/DEN4Δ30 parent or TBEV/DEN4Δ30/E-K₃₁₅D virus and attained peak viral titers six days later (FIG. 2A). TBEV/DEN4Δ30/E-K₃₁₅D virus was slightly less attenuated in this age of mice, demonstrating approximately a 10-fold decrease in replication compared to the parental virus.

When the mutant viruses were inoculated ic into brain of 5-day-old mice, they replicated to lower viral titers and attained peak viral titers later than the TBEV/DEN4Δ30 parent. TBEV/DEN4Δ30/E-K₃₁₅D and TBEV/DEN4Δ30/NS5-DR_(654,655)AA viruses were similar to each other, as they replicated between 25- and 32-fold less than the TBEV/DEN4Δ30 parental virus, respectively (FIG. 2B). However, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA virus was highly attenuated, as it replicated 20,000-fold less than TBEV/DEN4Δ30 virus, and attained its peak viral titer 12 days later (FIG. 2B). Sequence analysis of mouse brain isolates demonstrated that all of the designed mutations (E-K₃₁₅D, NS5-DR_(654,655)AA, and E-K₃₁₅D/NS5-DR_(654,655)AA) that were introduced in the chimeric genome were highly stable throughout the time course of the study in 5-day old mice (Table 5).

TABLE 5 Genetic stability of introduced mutations in TBEV/DEN4Δ30 viruses in brains of 5-day-old mice No. mutations Day of No. changed/No. tested^(c) Virus^(a) isolation^(b) tested Δ30 E₃₁₅ NS5_(654,655) TBEV/DEN4Δ30/E-K₃₁₅D  8 10  0/10  0/10 —  9  2 0/2 0/2 — TBEV/DEN4Δ30/NS5- 13  7 0/7 — 0/7 DR_(654,655)AA TBEV/DEN4Δ30/E- 15  2 0/2 0/2 0/2 K₃₁₅D/NS5-DR_(654,655)AA 17  4 0/4 0/4 0/4 ^(a)Five-day-old mice were inoculated IC with 10³ PFU of indicated virus. ^(b)Brains of mice were harvested on indicated day, and virus RNA was isolated from brain homogenate to determine virus genomic sequence. ^(c)The virus genome regions encompassing the introduced Δ30, E₃₁₅, or NS5_(654,655) mutations were directly sequenced from brain homogenates to determine stability of the mutations. Dashed lines indicate that no mutation was originally introduced at this position.

In addition to highly reduced neurovirulence in suckling mice, as shown by H&E staining, TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA did not cause neuroinflammation in the brains of mice inoculated intracerebrally, unlike TBEV/DEN4 and TBEV/DEN4Δ30 (FIG. 3).

Since the parental TBEV/DEN4 virus is highly attenuated for the ability to travel to the CNS by the ip route in immunocompetent mice (Pletnev et al., J. Virol. 67:4956-4963, 1993), the neuroinvasive properties of the modified TBEV/DEN4 viruses was investigated in immunocompromised mice. Groups of at least 10 SCID mice were ip inoculated with 10⁵ pfu of chimeric virus and AST, morbidity, and viral titers in the brains were assessed. Although introduction of the Δ30 deletion into the TBEV/DEN4 backbone demonstrated little effect on mouse neurovirulence, a decrease in mouse neuroinvasiveness was observed with TBEV/DEN4Δ30 virus, as shown by a reduction of virus-induced encephalitis or mortality (from 60% to 18%) compared to TBEV/DEN4 virus (Table 6). Since addition of the Δ30 mutation significantly reduced TBEV/DEN4 neuroinvasion in immunocompromised mice, the ability of Δ30 in combination with E-K₃₁₅D and/or _(NS)5-DR_(654,655)AA substitutions to further reduce this property was investigated. While the combination of E-K₃₁₅D with Δ30 increased the AST and decreased morbidity, introduction of both NS5-DR_(654,655)AA and Δ30 in TBEV/DEN4 completely abrogated the ability of the chimeric virus to invade the CNS from the peripheral inoculation site (Table 6). Infectious TBEV/DEN4Δ30/E-K₃₁₅D, TBEV/DEN4Δ30/NS5-DR₆₅₄₋₆₅₅AA, or TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA virus could not be recovered from brains of mice that were harvested at 13, 15, or 17 dpi, days at which the unmodified TBEV/DEN4 virus reached a peak virus replication in brains of SCID mice inoculated ic (FIG. 4). These studies demonstrate the neurological safety profile of TBEV/DEN4Δ30/E-K₃₁₅D, TBEV/DEN4Δ30/NS5-DR₆₅₄₋₆₅₅AA, and TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA virus.

TABLE 6 Neuroinvasiveness of chimeric viruses in SCID adult mice Dose % Virus (pfu) AST mortality TBEV/DEN4 10⁵ 23 60 TBEV/DEN4Δ30 10⁵ 22 18 TBEV/DEN4Δ30/E-K₃₁₅D 10⁵ 32 9 TBEV/DEN4Δ30/NS5-DR₆₅₄₋₆₅₅AA 10⁵ >49 0 TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR₆₅₄₋₆₅₅AA 10⁵ >49 0

Example 3 Demonstration of Immunoprotective Effect of TBEV/DEN4 Chimeras in Mice

This example demonstrates that TBEV/DEN4 chimeras are immunoprotective for TBEV/DEN4 infection in mice.

Methods

In the first experiment, adult Swiss mice were ip inoculated with 10⁵ pfu of TBEV/DEN4, TBEV/DEN4Δ30, or their respective mutant viruses and challenged via the ic route with 100 icLD₅₀ of TBEV/DEN4 28 days later. In the second experiment, six groups of 10 3-week-old Swiss mice were ip inoculated with one or two doses of 10⁵ pfu of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA or with three doses of the inactivated TBEV vaccine, Encepur® (30 μl per dose, or approximately one mouse protective dose). Three groups of mice were inoculated with two doses of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA on 0 and 21 dpi, whereas another three groups of mice were administered one dose of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA on 21 dpi. The remaining three groups of mice were inoculated with three doses of Encepur® on 0, 7, and 21 dpi, in a schedule comparable to the rapid vaccination schedule in humans. An additional group of 20 mice were ip inoculated with Leibovitz's L-15 medium (Invitrogen) and separated into challenge control groups. On day 48 post-inoculation, all mice were ip inoculated with 100 LD₅₀ (10² pfu) of homologous TBEV strain Sofjin, or heterologous TBEV strain Hypr.

In both sets of experiments, serum was measured for neutralizing antibody titers by the plaque reduction neutralization test (PRNT) 25 days following the last immunization. Briefly, 4-fold serially diluted heat-inactivated sera were combined with TBEV/DEN4Δ30 virus and 10% guinea pig complement (Lonza Inc., Allendale, N.J.), incubated for 1 hour at 37° C., and then added to monolayers of Vero cells in two replicates. Antibody titers were defined as the dilution of serum that neutralized 60% of the virus (PRNT₆₀). Seroconversion was defined as a 4-fold increase in serum neutralizing antibody titers compared to the negative controls.

All mice were monitored for signs of morbidity and mortality for 21 days post-challenge (dpc). Moribund mice were euthanized upon observation of symptoms, including paralysis and hemorrhaging. Average survival times (ASTs) were determined by measuring mean survival time of mice that succumbed to infection within 21 dpc.

Results

In the first experiment, all control mice succumbed to infection by day 9 post-inoculation following a severe is challenge with TBEV/DEN4 virus. However, TBEV/DEN4 and TBEV/DEN4/E-K₃₁₅D inoculated mice were completely protected against challenge with the lethal dose of TBEV/DEN4 virus, while TBEV/DEN4/NS5-DR_(654,655)AA and TBEV/DEN4/E-K₃₁₅D/NS5-DR_(654,655)AA viruses induced between 40% and 60% protection for the mice (FIG. 5A). Furthermore, 80% of TBEV/DEN4Δ30 inoculated mice were protected against severe challenge with TBEV/DEN4 virus, while viruses containing E-K₃₁₅D, NS5-DR_(654,655)AA, or both substitutions within the TBEV/DEN4Δ30 backbone also provided protection in 40-60% of the mice (FIG. 5B). Mice inoculated with either TBEV/DEN4 or TBEV/DEN4Δ30 mutants exhibited slight delays in death compared to the control groups (FIGS. 5A and 5B). Although these studies demonstrated moderate protection from severe challenge with TBEV/DEN4, neutralizing antibody titers in all groups of mice were low (<1:20 geometric mean titer) but were similar to each other.

In the second experiment, adult Swiss mice inoculated intraperitoneally also showed that two doses (10⁵ pfu) of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA induced (1) comparable levels of TBEV-specific serum neutralizing antibodies to three doses of the inactivated Encepur® vaccine (Novartis) (FIG. 6). Furthermore, similar levels of animals seroconverted when vaccinated either with two doses of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA or with three doses of Encepur® (50% vs, 68% respectively. Two doses of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA also demonstrated similar levels of protection compared to three doses of Encepur® after challenge with wild-type homologous TBEV strain Sofjin or heterologous TBEV strain Hypr viruses (Table 7). Overall, these studies demonstrate that the immunogenicity and/or protection of TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA is comparable to that of the inactivated Encepur® vaccine in mice.

TABLE 7 Protection of inoculated mice challenged with wild-type TBEV strains No. No. Challenge % Immunizing virus^(a) doses animals virus^(b) Survival AST^(d) TBEV/DEN4Δ30/E- 1 10 Sofjin 10   9.9 K₃₁₅D/NS5-DR_(654,655)AA 2 10 60^(§)  9.8 Encepur ® 3 10 80^(§) 17.0 Mock  5  0  10.0 TBEV/DEN4Δ30/E- 1 10 Hypr  0  10.8 K₃₁₅D/NS5-DR_(654,655)AA 2 10 70^(§) 12.3 Encepur ® 3 10 80^(§) 11.0 Mock  5  0  10.8 ^(a)Groups of mice were inoculated with one or two doses of 10⁵ pfu TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA virus, or three doses of inactivated Encepur ® vaccine. ^(b)Mice were challenged with 100 ip LD₅₀ of wild-type viruses on day 48 pi. ^(c)Seroconversion was defined as 4-fold increase in neutralizing antibody titer compared to original dilution. ^(d)Average survival times of mice that succumbed to disease following challenge with wild-type TBEV. ^(§)Significantly different from other groups (Kaplan-Meier, log-rank p < 0.05), but not from each other. 50% animals seroconverted with 2 doses of LA vaccine, 68% animals seroconverted with 3 doses of Encepur ® (no significant difference between the two groups)

Example 4 Demonstration of Protective Effect of Chimeric Viruses in Rhesus Macaques

This example demonstrates that the chimeric TBEV/DEN4 viruses are immunogenic in rhesus macaques and provide a protective effect following challenge.

Methods

The studies in monkeys with the chimeric TBEV vaccine candidates were carried out in the BSL-3 facility at Bioqual, Inc. (Rockville, Md.), in accordance with all state and federal guidelines. All monkeys were seronegative for neutralizing antibodies to TBEV or DEN4 prior to immunization. Groups of 4 rhesus macaques (Macaca mulatta) were immunized subcutaneously (sc) with one dose of 10⁵ pfu of chimeric TBEV/DEN4Δ30 virus or its derivatives. One group of 4 monkeys received 3 human doses of an inactivated TBEV vaccine (ENCEPUR®, Chiron/Behring, Germany) in an immunization schedule that is similarly used for humans. One dose was administered on day 0, and monkeys were boosted with inactivated TBEV vaccine on days 7 and 21. Monkeys inoculated with live virus were bled daily under ketamine anesthesia for 9 days to detect and quantitate levels of viremia. Blood was collected from monkeys on 42 dpi to measure levels of TBEV-specific neutralizing antibodies against TBEV/DEN4Δ30, wild-type Far-Eastern TBEV subtype Sofjin, or wild-type Central European TBEV subtype Hypr. All monkeys were challenged sc with 10⁵ pfu of unmodified TBEV/DEN4 virus the following day. Monkeys were bled daily on days 0 to 7 post-challenge (43-50 dpi) to determine levels of viremia and on 21 dpc (64 dpi) for measurement of serum neutralizing antibodies against TBEV/DEN4Δ30. PRNT₆₀ assays against TBEV/DEN4Δ30 were done according to the protocol described in Example 3, whereas PRNT₆₀ assays against wild-type TBEV were performed using 4-fold serially diluted sera combined with either Sofjin or Hypr virus. The serum:virus mix was incubated at 37° C. for one hour, and then added to confluent monolayers of BHK cells in replicates of two.

Results

Since the TBEV/DEN4Δ30 mutant viruses demonstrated the lowest values for neurovirulence and neuroinvasion compared to TBEV/DEN4 and still provided protection in mice against severe challenge with TBEV/DEN4 virus, these viruses were analyzed for safety, immunogenicity, and protective efficacy in non-human primates. Analysis was performed by investigating levels of viremia and TBEV-specific neutralizing antibody titers, since non-human primates do not exhibit any disease manifestation after peripheral inoculation with unmodified TBEV/DEN4 virus (Rumyantsev et al., Vaccine 26:133-143, 2006). Rhesus macaques were sc inoculated with one dose of TBEV/DEN4Δ30 virus or its derivatives, or three doses of the inactivated TBEV vaccine, bled to detect levels of viremia and neutralizing antibody titers, and sc challenged with unmodified TBEV/DEN4 virus. All animals were healthy throughout the study, regardless of the inoculums used.

One animal within the TBEV/DEN4Δ30 group and two animals in the TBEV/DEN4Δ30/E-K₃₁₅D vaccinated groups exhibited low levels of viremia (≦1.5 log₁₀ pfu/ml) for one to two days, while all remaining monkeys demonstrated no viremia above detectable limits (Table 8). This was in contrast to animals inoculated with TBEV/DEN4 virus, which developed viremia that lasted three to four days and attained a mean peak virus titer of 3.1 log₁₀ pfu/ml. Despite low to no levels of viremia, all animals that received a single dose of any of the modified TBEV/DEN4 viruses seroconverted and had mean TBEV neutralizing antibody titers between 65 to 150, while animals inoculated with either the parental TBEV/DEN4 virus or three doses of the inactivated TBEV vaccine demonstrated higher mean neutralizing antibody titers of 1817 and 899, respectively (Table 8).

Although the modified TBEV/DEN4 viruses induced 6-14-fold lower mean neutralizing titers compared to three doses of the inactivated vaccine, they were still able to provide protection against viremia caused by challenge with TBEV/DEN4 virus. However, mean TBE neutralizing antibody titers after vaccination with any of the TBEV/DEN4Δ30 mutant viruses increased post-challenge, demonstrating the inability of these viruses to completely prevent viral replication after challenge.

TABLE 8 Viremia and serum neutralizing antibodies in rhesus monkeys in response to TBEV/DEN4 chimeras Viremia (log₁₀ pfu/ml) on Serum Viremia (log₁₀ pfu/ml) on Serum Serum Immunizing Rhesus indicated day post-inoculation^(a) neutr. titer indicated day post-challenge^(c) neutr. titer neutr. titer virus No. 0 1 2 3 4 42 dpi^(b) 0 1 2 3 4 64 dpi 84 dpi TBE/DEN4Δ30 DC25 — — — — — 120 — — — — — 2175 880 DC44 — — 1.4 — — 203 — — — — — 764 286 DC53 — — — — — 135 — — — — — 1137 465 DC62 — — — — — 152 — — — — — 1623 1449 GMT^(b) 150 1323 642 TBE/DEN4Δ30/ DC72 — — 1.0 — — 175 — — — — — 454 270 E-K315D DC81 — 0.7 1.5 — — 121 — — — — — 363 301 DC1L — — — — — 40 — — — — — 826 679 DC2J — — — — — 52 — — — — — 3692 1663 GMT 82 842 550 TBE/DEN4Δ30/ DCR1 97 1795 661 NS5-DR_(654, 655)AA FIC — — — — — 61 — — — — — 1014 613 FID — — — — — 81 — — — — — 284 317 BZH — — — — — 53 — — — — — 453 223 GMT 71 696 411 TBE/DEN4Δ30/ F78 65 961 457 E-K315D/NS5- DR_(654, 655)AA clone A DBV3 — — — — — 102 — — — — — 519 221 DBVF — — — — — 17 — — — — — 776 125 DBVW — — — — — 152 — — — — — 2052 574 GMT 65 944 292 Inactivated C70 624 2407 1718 TBEV vaccine (3 doses) DBW8 — — — — — 400 — — — — — 413 387 DBPD — — — — — 2399 — — — — — 1537 1670 DBPF — — — — — 1090 — — — — — 456 96 GMT 899 914 571 Mock DB1N — — — — — <2 — 2.7 2.3 1.7 1.0 1136 4374 DBRI — — — — — <2 — 1.8 3.3 1.0 — 1287 3380 DBC7 — — — — — <2 — — 0.7 1.8 — 5946 4262 A5E071 — — — — — <2 — — 2.3 — — 1254 1652 GMT <2 1817 3194 ^(a)Dashed lines (—) indicate serum viral titers below the limit of detection (<0.7 log₁₀ pfu/ml) in Vero cells. Serum viral titers were measured 0-8 days post-immunization or 0-7 days post-challenge. Viral titers in all groups were below the limit of detection 3-8 days post-immunization and 5-7 days post-challenge and are not shown. ^(b)Plaque reduction (60%) neutralizing antibody titer was determined against TBEV/DEN4Δ30 virus using serum collected on indicated day post-immunization. Reciprocal titers are shown; geometric titers (GMT) are calculated for each group. ^(c)Monkeys were challenged sc with 10⁵ pfu of TBEV/DEN4 virus 43 days post-immunization.

Serum neutralizing antibody titers in rhesus monkeys against Sofjin and Hypr viruses were similar between groups of monkeys inoculated with one dose (10⁵ pfu) of TBE/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA and three doses of Encepur® vaccine (Table 9). Overall these studies demonstrate that immunogenicity and/or protection by TBE/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA is comparable to that of the inactivated Encepur® vaccine in monkeys.

TABLE 9 Immunogenicity of rhesus monkeys against homologous or heterologous wild-type TBEV following vaccination Serum neutralizing Rhesus antibody titer^(b) Immunizing virus^(a) no. α-Sofjin α-Hypr TBE/DEN4Δ30 DC25 17 11 DC44 79 58 DC53 22 26 DC62 21 32 GMT 28 27 TBE/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA F78 7 19 DBV3 37 <5 DBVF 13 <5 DBVW 46 93 GMT 20 13 Encepur C70 11 27 3 doses DBW8 12 46 DBPD 52 273 DBPF 37 105 GMT 22 77 ^(a)Groups of four monkeys were inoculated SC with one dose of 5.0 log₁₀ PFU of TBEV/DEN4Δ30 or TBEV/DEN4Δ30 Δ30/E₃₁₅/NS5_(654,655) , or inoculated sc with three doses of 0.5 ml of Encepur ® vaccine. ^(b)Serum was harvested from monkeys on day 42 pi, prior to TBEV/DEN4 virus challenge. Serum plaque reduction (60%) neutralizing antibody titers were determined against wild-type Far-Eastern strain Sofjin or Central European strain Hypr. Reciprocal titers are shown; geometric mean titers (GMT) are calculated for each group. No significant differences were observed between serum neutralizing antibody titer groups (p > 0.05, one-way ANOVA).

Example 5 Virus Infection in Mosquitoes and Ticks

This Example demonstrates the inability of the TBEV/DEN4Δ30 chimeric viruses to infect and replicate mosquitoes and ticks.

Methods

Virus infection in mosquito and tick cell culture. To investigate the viral kinetics in cell culture, confluent monolayers of Vero or Aedes albopictus C6/36 cells were infected at a multiplicity of infection (MOI) of 1 for 1 hr at 37° C. or 32° C., respectively, and maintenance media was added. Virus supernatant was harvested from the cell culture every 24 hr for 8 days and frozen at −80° C. until it could be titrated by immunofocus assay. After harvesting the supernatant, fresh media was added. To determine viral titers, confluent monolayers of Vero cells in 24- or 48-well plates were infected with 10-fold serial dilutions of virus, incubated at 37° C. for 1 hr, and were overlaid with Opti-MEM I containing 1% methylcellulose (Invitrogen), 2% heat-inactivated FBS, 4 mM L-glutamine, and 0.05 mg/ml of gentamycin. After incubation for 6 days at 32° C., the cells were fixed for 20 min with 100% methanol, and plaques were visualized by immunostaining with hyperimmune mouse ascitic fluid specific for both TBEV and LGT virus (Russian Spring Summer Encephalitis (RSSE) VR79; ATCC) and peroxidase-labeled polymer conjugated to anti-mouse immunoglobulin (Dako Co., Carpinteria, Calif.). Mean viral titers from each time point were determined from three replicates.

To investigate the ability of the viruses to infect and replicate in tick cells, infection of Ixodes scapularis ISE6 cells was undertaken. Infection of simian Vero cells was used as a positive control. For ISE6 cells, all viruses were serially passaged three times following an initial infection of ISE6 cells with virus. Confluent monolayers of ISE6 tick or Vero cells were initially infected with virus at a MOI of 1, and cells were incubated at 34° C. or 37° C., respectively, for 1 hr. Maintenance media was added following incubation and ⅓ volume of the virus supernatant was obtained from infected cells after 7 days; the supernatant was then used to infect fresh ISE6 cells. The infected cells were stained for viral antigen by immunofluorescence or used to extract viral RNA following all three passages with virus.

Detection of viral antigen in cell culture. After 5 days (Vero cells) or 7 days (ISE6 cells), cells were assayed for infection by immunofluorescence. Slides were prepared by cytocentrifugation of 5×10⁴ cells followed by fixation in 100% acetone for 30 min. The slides were blocked with PBS containing 2% normal goat serum (NGS) and 1% bovine serum albumin (BSA) for 30 min at room temperature. The primary antibody was a cocktail containing the polyclonal mouse antibody cross-reactive to TBEV and LGT virus (RSSE), and DEN4 virus antiserum (hyperimmune mouse ascites fluid, HMAF). Both antibodies were diluted at 1:1000 in blocking buffer. Following application of the primary antibody, the slides were incubated for 1 hr at 37° C. and were washed with PBS containing 0.5% Tween-20 (PBS-T). Secondary antibody (goat anti-mouse IgG conjugated with Alexa Fluor® 488 (Invitrogen)) was applied at a 1:500 dilution in blocking buffer and incubated for 1 hr at 37° C. The slides were washed in PBS-T and coverslips were mounted using ProLong® Gold antifade reagent (Invitrogen) with 4′6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were captured using an Olympus BX51 microscope with an Olympus DP70 camera and Microsuite software.

Virus infection in mosquitoes. Aedes aegypti mosquitoes were reared at 27° C. in 70% relative humidity with a 16-hr daylight cycle for use in oral infection. For oral infection of mosquitoes, 10⁶ pfu/ml of each virus was mixed separately with defibrinated rabbit red blood cells (Spring Valley Laboratories, Woodline, Md.) containing 2.5% sucrose. Five-day-old female Ae. aegypti mosquitoes that had been deprived of a sugar source for 24 hr were exposed to the virus bloodmeal for 25 min. The infected bloodmeal was prepared immediately prior to feeding and was offered to mosquitoes in a 37° C. preheated water jacketed feeder covered in stretched Parafilm®. Fully engorged mosquitoes were transferred to a new container by aspirator and maintained as described above for either 14 or 21 days post-infection (dpi). Throughout the study, the mosquitoes were allowed continuous access to a cotton pad soaked in a solution of 10% sucrose. After 14 or 21 days, mosquitoes were harvested and stored at −80° C. until dissection.

To assess virus infection of mosquitoes, the bodies of mosquitoes were first separated from the legs and heads and then separately triturated in 250 μl Hanks balanced salt solution (HBSS) (Invitrogen) supplemented with 10% FBS, 250 μg/ml amphotericin, 1% ciprofloxacin, and 150 mg/ml clindamycin. To address the efficiency of viral dissemination, individual mosquito heads were also triturated in 250 μl HBSS containing FBS, amphotericin, ciprofloxacin, and clindamycin. Virus titers of heads and bodies were determined in Vero cells by immunofocus assay, as described above.

Virus infection in ticks. I. scapularis adult females with egg sacs (Oklahoma State University, Stillwater, Okla.) were housed in a relative humidity of 98% with a 16-hr daylight cycle for oviposition and larvae emergence. The larvae were used within 6 months of emergence.

Infection of ticks was by the immersion method. Briefly, 60 larvae per group were collected in a sterile 1.5 ml screw cap centrifuge tube and pretreated by exposure to a reduced relative humidity. To virally infect ticks, the larvae were then immersed in 0.5 ml of complete medium containing 10⁶ pfu/ml of virus and incubated at 34° C. for 45 min. Following immersion, the ticks were washed twice with cold PBS. Larvae were wicked free of excess moisture using tapered strips of Whatman® paper and maintained at a relative humidity of 98% for 21 and 45 days post-immersion (dpim).

Detection of viral RNA from cell culture or whole ticks. Total RNA was isolated from Vero and ISE6 cells using the RNeasy® mini kit (Qiagen, Valencia, Calif.). Cell pellets were resuspended in 350 μl of Buffer RLT and were homogenized by passing through a QIAshredder spin column (Qiagen). RNA was eluted in 50 μl of RNase-free water.

To isolate total RNA from ticks, a group of 25 ticks were homogenized using one of two methods. In the first method, ticks were frozen in liquid nitrogen and triturated with mortar and pestle. The powder was resuspended in 250 μl of Buffer RLT and passed through a QIAshredder spin column. For the second method, ticks were frozen in liquid nitrogen and transferred to Lysing Matrix D tubes (MPBio, Solon, Ohio) containing 800 μl of RLT buffer (Qiagen RNeasy® kit). The ticks were homogenized using the Fastprep® 24 (MPBio) set at speed level 6 m/s for 40 s. The homogenate was clarified by centrifugation at 21,000×g for 3 min. For both methods, RNA from the homogenates was purified using an RNeasy® mini kit (Qiagen). The RNA was eluted in 35 μl of RNase-free water.

Viral RNA from cell culture and ticks was detected using SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen) and primers that were specific for positive- or negative-sense RNA. The RNA was first reversed transcribed at 55° C. for 30 min, followed by routine PCR conditions (40 cycles of denaturing at 94° C. for 1 min, annealing at 55-69° C. for 45 s, and extending at 68° C. for 3 min). The primers used in the reactions are listed in Table 10, and were specific to DEN4, TBE (Sofjin), LGT, or LGT/DEN4 only. The PCR amplicons were examined on a precast 1.2% agarose gel (Lonza).

TABLE 10 Primers used for RT-PCR of negative- and positive-sense RNA SEQ Virus Primer Sequence ID DEN4 5′ Primer CCAGAGTCCCCAGCGAGACTAG 13 (+ Sense Strand)^(a) 3′ Primer GCCAAGGGGTAGAGACCTGAC 14 DEN4 5′ Primer CTCCATGACGCCACACAACCCATGTC 15 (− Sense Strand)^(a) 3′ Primer CTCAGAAACCCAGGATTCGCGCTCTTGG 16 TBE 5′ Primer GCCACAGTGCGGAAGGAAAGAG 17 (+ Sense Strand)^(b) 3′ Primer GGATCTTGGGCAAGAACCCCACTC 18 TBE 5′ Primer CACCGCCAAGAACTGTGTGCA 19 (− Sense Strand)^(b) 3′ Primer GACCGTGGAAAGTGTGGTGAC 20 LGT 5′ Primer CAGCGACTGTGATTGTGGATATTC 21 (+ Sense Strand)^(c) 3′ Primer AAGGTTGGGTTCCTCATGTTCAAGC 22 LGT 5′ Primer ACTGGCCGGTAGAAACAGCTT 23 (− Sense Strand)^(c,d) 3′ Primer AAGGTTGGGTTCCTCATGTTCAAGC 24 LGT/DEN 5′ Primer CGCTCCTCCCAGGACGGTGTGC 25 (− Sense Strand)^(c,e) 3′ Primer GCGTCGAGATGCACCCACCTGGA 26 ^(a)Sequence of DEN4 vector p4 (Genbank Accession No. AY648301) ^(b)Sequence of TBEV Sofjin strain (Genbank Accession No.X07755) ^(c)Sequence of LGTV TP21 strain (Genbank Accession No. AF253419) ^(d)Negative-sense primer used to detect LGTV infection ^(e)Negative-sense primer used to detect LGTV/DEN4 infection Results

The ability of TBEV/DEN4Δ30 chimeric viruses to infect and replicate in tick and mosquito cell culture, as well as Ixodes scapularis ticks and Aedes aegypti mosquitoes (arthropod vectors that are able to transmit TBEV and DEN4 virus, respectively) was investigated. Although the chimeric viruses were able to moderately replicate in mosquito cell culture (FIG. 7), they were unable to infect, replicate, or disseminate in Ae. aegypti mosquitoes (Table 11, FIG. 8). In addition, the chimeric viruses were unable to infect tick cells, as shown by absence of viral antigen and absence of virus RNA following passage in cells (Table 12). Furthermore, the chimeric viruses were unable to infect or replicate in I. scapularis ticks (FIG. 9). These studies demonstrate the environmental safety profile of the vaccine candidates, including TBEV/DEN4Δ30/E-K₃₁₅D/NS5-DR_(654,655)AA.

TABLE 11 Infection and dissemination rates of TBEV/DEN4Δ30 chimeras, DEN4, and LGT viruses in mosquitoes 14 dpi 21 dpi No. No. No. dissem- No. dissem- infected inated infected inated (% (% (% (% Virus^(a) positive)^(b) positive)^(c) positive)^(b) positive)^(c) DEN4 16/18 (89) 13/16 (81) 15/18 (83) 15/15 (100) TBEV/DEN4Δ30 0/18 (0) 0/18 (0) 0/18 (0) 0/18 (0)  TBEV/DEN4Δ30/E- 0/18 (0) 0/18 (0) 0/18 (0) 0/18 (0)  K₃₁₅D TBEV/DEN4Δ30/NS5- 0/18 (0) 0/18 (0) 0/18 (0) 0/18 (0)  DR_(654,655)AA TBEV/DEN4Δ30/E- 0/18 (0) 0/18 (0) 0/18 (0) 0/18 (0)  K₃₁₅D/NS5- DR_(654,655)AA LGT 0/18 (0) 0/18 (0) 0/18 (0) 0/18 (0)  ^(a) Ae. aegypti mosquitoes were orally infected with the indicated viruses and incubated for 14 or 21 dpi. Mosquito bodies and heads were separated, triturated, and titered in Vero cells to determine infectivity and dissemination rates. ^(b)Infection rates were measured by the presence of virus in the bodies of mosquitoes compared to the number of mosquitoes tested. ^(c)Dissemination rates were measured by the presence of virus in the heads of mosquitoes compared to the number of mosquitoes tested.

TABLE 12 Detection of viral RNA in ISE6 cells during three serial passages Passage 1^(a) Passage 2^(b) Passage 3^(c) Posi- Nega- Posi- Nega- Posi- Nega- Virus tive^(b) tive^(b) tive^(b) tive^(b) tive^(b) tive^(b) LGT + + + + + + DEN4 + − − − − − TBEV/DEN4Δ30 + + − − − − TBEV/DEN4Δ30/E- + + − − − − K₃₁₅D TBEV/DEN4Δ30/ + + − − − − NS5-DR_(654, 655)AA TBEV/DEN4Δ30/E- − − − − − − K₃₁₅D/NS5- DR_(654, 655)AA ^(a)Presence and absence of viral RNA is denoted by + or −, respectively. ^(b)Positive- or negative-sense RNA detection by RT-PCR.

Example 6 Vaccination of Human Subjects with Recombinant TBEV/DEN4Δ30 Chimeric Viruses

This example describes evaluation of candidate TBEV/DEN4 chimeric virus vaccines in human subjects.

The safety and efficacy of the disclosed recombinant TBEV/DEN4 chimeras can be evaluated in human volunteers according to procedures well known in the art (e.g., Wright et al., Vaccine, 26:882-890, 2008). For example, eligibility criteria can include: age 18-50 years; no history of chronic illness; normal findings during physical examination; negative (<1:10) for serum neutralizing antibodies to Powassan, Langat, TBEV, West Nile virus, dengue virus types 1-4, and yellow fever virus; negative for antibodies to Saint Louis encephalitis virus and Japanese encephalitis virus by hemagglutination-inhibition (titer <1:10); negative human immunodeficiency virus antibody test and hepatitis C virus antibody test; negative for hepatitis B surface antigen; normal hematologic and hepatic values; and normal urinalysis. Female volunteers should have a negative result on a urine pregnancy test prior to vaccinations and on the days of vaccination and agree to use contraception or abstain from sexual intercourse for the duration of the study. Human volunteers may be selected from those at risk of infection with TBEV, such as individuals residing in areas where TBEV is endemic.

In this example, human volunteers are injected with candidate TBEV/DEN4Δ30 chimeric vaccines subcutaneously at an appropriate dose. The appropriate dose is the dose approved by the FDA, and can be determined from suitable animal studies conducted prior to human vaccination trials (such as those described in Examples 2 and 3). Other routes of administration are possible, including intramuscular and intravenous. The vaccine can be administered as a single dose, or given in multiple doses, such as two, three or four doses. When administered in multiple doses, the booster doses can be administered at various time intervals, such as months to years. Serum samples can be obtained to determine neutralizing antibody titers and identify responders and non-responders to the vaccine.

The initial phase of the study is a double-blind placebo controlled trial in which volunteers are randomly assigned to receive the vaccine or placebo (e.g., in a 4:1 ratio). Volunteers are enrolled in a step-wise manner: a first set of volunteers (such as 3-5 subjects) are initially enrolled; after study day 21, all safety data is reviewed by the medical monitor before proceeding with enrollment of the next set of volunteers (such as about 8-12 subjects); and the set of safety data collected for these volunteers is then reviewed by the medical monitor before enrollment of the remaining volunteers (such as 12-20 subjects). Randomization is done such that no more than 1 volunteer in the first group of volunteers and no more than 4 volunteers in the second group of volunteers receive placebo. Volunteers are observed 30 min after vaccination for any immediate reaction to the vaccine. They are given a diary card and a digital oral thermometer to record their temperature 3 times daily for 19 days. Volunteers return to the clinic every other day for 16 days after vaccination and on study days 19, 21, 28, 42, and 180. A clinical assessment is done at each visit and blood is obtained for hematological and clinical chemistry testing and for virologic or immunologic analysis. Serum is titrated for vaccine virus and for neutralizing antibody.

After completion of initial vaccination, the study may be modified to include a second dose to assess the response of vaccinees to a booster dose. Six to 18 months after initial vaccination the volunteers are re-randomized to receive either vaccine or the placebo control (vaccine diluent) (Wright et al., Vaccine, 26:882-90, 2008; McArthur et al., Am. J. Trop. Med. Hyg. 79:678-84, 2008; Durbin et al., Hum Vaccin., 2:255-60, 2006; Durbin et al., Hum Vaccin., 2:167-73, 2006; Durbin et al., J Infect Dis., 191:710-8, 2005).

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. 

We claim:
 1. A recombinant chimeric virus comprising: a first nucleic acid molecule comprising: a 5′ non-coding region from a dengue type 4 virus; a nucleic acid encoding: a C protein from a dengue type 4 virus; and non-structural proteins from a dengue type 4 virus, wherein nonstructural protein NS4B comprises a phenylalanine at amino acid position 112, and nonstructural protein NS5 comprises an alanine at amino acid position 654 and an alanine at amino acid position 655; and a 3′ non-coding region from a dengue type 4 virus, wherein the 3′ non-coding region comprises a deletion of nucleotides 10478-10507; and a second nucleic acid molecule operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding: a prM protein from a tick borne encephalitis virus; and an E protein from a tick borne encephalitis virus, wherein the E protein comprises an amino acid substitution from wild-type at amino acid position 315 and a tryptophan at amino acid position
 240. 2. The recombinant chimeric virus of claim 1, wherein the amino acid substitution at amino acid position 315 of the E protein is selected from the group consisting of aspartic acid, alanine, phenylalanine, leucine, serine, arginine, threonine, tryptophan, valine, and tyrosine.
 3. The recombinant chimeric virus of claim 1, further comprising an amino acid substitution at amino acid position 84 of the E protein.
 4. The recombinant chimeric virus of claim 1, further comprising an amino acid substitution at amino acid position 6 of nonstructural protein NS1.
 5. The recombinant chimeric virus of claim 1, wherein the tick-borne encephalitis virus is selected from the group consisting of Central European, Siberian, and Far Eastern subtype.
 6. The recombinant chimeric virus of claim 1, wherein the virus encodes a polypeptide with at least 95% identity to SEQ ID NO:
 2. 7. The recombinant chimeric virus of claim 6, wherein the virus encodes a polypeptide comprising SEQ ID NO:
 2. 8. The recombinant chimeric virus of claim 7, wherein the virus encodes a polypeptide consisting of SEQ ID NO:
 2. 9. The recombinant chimeric virus of claim 1, wherein the virus comprises a nucleic acid molecule with at least 95% identity to SEQ ID NO:
 1. 10. The recombinant chimeric virus of claim 9, wherein the virus comprises a nucleic acid molecule comprising SEQ ID NO:
 1. 11. The recombinant chimeric virus of claim 10, wherein the virus comprises a nucleic acid molecule consisting of SEQ ID NO:
 1. 12. A method of eliciting an immune response to tick-borne encephalitis virus in a subject, comprising administering a therapeutically effective amount of the recombinant chimeric virus of claim 1 to the subject.
 13. The method of claim 12, further comprising selecting a subject in need of enhanced immunity to tick-borne encephalitis virus.
 14. An immunogenic composition comprising the recombinant chimeric virus of claim 1 and a pharmaceutically acceptable carrier.
 15. A method of eliciting an immune response to tick-borne encephalitis virus in a subject, comprising administering to a subject a therapeutically effective amount of the immunogenic composition of claim
 14. 16. A method of eliciting an immune response to a member of the tick-borne encephalitis virus complex in a subject, comprising administering the recombinant chimeric virus of claim 1 to the subject.
 17. The method of claim 16, wherein the member of the tick-borne encephalitis virus complex is Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Langat virus, Louping ill virus, Negishi virus, or Powassan virus.
 18. The method of claim 15, further comprising administering to the subject an immunogenic composition for one or more additional flaviviruses. 